tag:blogger.com,1999:blog-80377073523304287882024-03-08T15:15:31.819-06:00UCFlow - Flow Cytometry news, reviews, and tips.A Blog about the world of Image and Flow Cytometry. Coming to you from the core facility at the University of ChicagoUC Flowhttp://www.blogger.com/profile/03187449850452376466noreply@blogger.comBlogger124125tag:blogger.com,1999:blog-8037707352330428788.post-40729469492219887512015-09-25T18:11:00.002-05:002015-09-25T18:12:57.685-05:00The NovoCyte Analyzer Review - Acea Biosciences<div dir="ltr" style="line-height: 1.38; margin-bottom: 0pt; margin-top: 0pt;">
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhBSlPJykGfh-m473dgdpruSqgEEuQoUDFnAbNhoixL_LvcHI6hzDXocNuSv6MX5YvPzCcZU2q_4FR8RZx77AyaOWo9IX8-167OqEi-uY0lTMSapLaS3v9T9FY-q06sD04o0nsZy3FYvmeB/s1600/NovoCyte_Industrial_Design_730.png" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="195" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhBSlPJykGfh-m473dgdpruSqgEEuQoUDFnAbNhoixL_LvcHI6hzDXocNuSv6MX5YvPzCcZU2q_4FR8RZx77AyaOWo9IX8-167OqEi-uY0lTMSapLaS3v9T9FY-q06sD04o0nsZy3FYvmeB/s400/NovoCyte_Industrial_Design_730.png" width="400" /></a></div>
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Why enter the crowded flow cytometer market with a 3-laser, 13-color analyzer? Why not? Acea Biosciences (San Diego, CA) is yet another upstart cytometry company with aspirations of creating an easy-to-use, affordable workhorse analyzer targeted at the meat of all flow cytometry experiments hovering in the 6-8 parameter range. The NovoCyte is a 3-laser, 13 fluorescent parameter analyzer ready to do battle with the likes of Industry giant FACSCanto-II (Becton-Dickinson), a few newcomers like the CytoFlex (Beckman-Coulter), the Attune NxT (Thermo-Fisher), and other perennial contenders such as the Miltenyi MACSQuant, and instruments from Stratedigm, and Partec/Sysmex. Of course, it's configurable starting at just 1 laser and 3 colors (1/3), with 2/4, 2/6, and 3/13 options (405nm, 488nm and 640nm). It’s pretty clear 3 lasers is the new 2, 4 lasers is the new 3, and 5+ lasers is the new 4. I had a chance to spend a couple of months with the NovoCyte and can say that the goal post for a quality cytometer has once again been moved back. Sure, it’s not in the same class spec wise, with the BD LSR-Fortessa, but then again, how many applications truly require a 5-laser 20 parameter, $350K instrument? Like most things in life, it’s all about the trade-off. Yes, there are some sacrifices you’ll need to make when going with the NovoCyte, but in many cases, it’s a small price to pay because, quite literally, it’s a small price to pay. </span></div>
<b id="docs-internal-guid-4a5da945-1d84-b831-bda8-53dd11ab2368" style="font-weight: normal;"><br /></b>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">No doubt, many readers are likely in the same position as me. I have BD instruments running FACSDiVa and a bunch of users who know DiVa inside and out. It’s also probably true that you have your favorite set of expletives you rattle off every time you use a BD instrument running FACSDiVa. The tyranny of the default is extremely powerful, and the only way to break through the FACSDiVa wall is to create a cytometer that is a pleasure to use. I always like to say there are only two parts of a cytometer with which an end-user interacts - the software, and the sample loading apparatus. If you can nail these two parts of the instrument, you’ll be able to win over a large part of the community. Conversely, if these components fail from a usability standpoint, you can have the best performing instrument out there, and gain no traction at all in the marketplace. The NovoCyte hits both of these key features pretty well. Their implementation of the autosampler is probably the best I’ve seen to date. It’s smooth, extremely flexible in the types of tubes/plates it accepts, it’s fast enough, and frankly, it just looks cool. In fact, one of the more impressive things about the NovoCyte in general, is its build quality. It's encapsulated in a sturdy metal skin that is completely white with a few touches of color here and there. The doors and cover are secure yet easy and smooth to open. Overall, it has a pretty small footprint, even when you add in the fluidics tray and the autosampler. </span></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><br /></span></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">The software takes a bit getting use to, but once you understand the workflow, it’s a breeze. You can easily pick up on the design cues implemented in their software - the structure of FACSDiVa, the drag-n-drop of FlowJo, and the ribbon layout of MS Office. It’s friendly enough that you won’t shy away immediately, but it will certainly take you a few minutes to grasp your bearings. The NovoCyte also excels at some other usability features that even surprised a cytometry veteran like me. I especially enjoyed the physical startup/shutdown button on the face of the instrument. This single button not only turns the cytometer on, but also performs the fluidics startup routine before you even turn the computer on. Likewise, when you’re rushing out the door in the evening and you need to shut down the instrument, just click the button once again and it will automagically perform the shutdown routine and power off the instrument. I can’t tell you how many times I’ve rushed through a shut-down procedure skipping as many steps as I could in order to get out the door on time. It’s clear a lot of effort and forethought went into many of these usability features. Is this enough to breakthrough the DiVaDefault</span><span style="background-color: transparent; color: black; font-family: Arial; font-size: 8.799999999999999px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: super; white-space: pre-wrap;">TM</span><span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">? I think it might have a chance, at least. </span></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: large; font-style: normal; font-variant: normal; vertical-align: baseline; white-space: pre-wrap;"><u>Fluidics</u></span></h3>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEieTZXtVGMcm3-UKO8Ut_1-aZPg23TBOOm8dBpa-uu17WWPxJgcHHoQpzvaimBUN3ZyuP2G69KTk01ojmCnFvd6sxAd-l5rluuPFeiCq1oE-yCy75SgtFAZwGLTfYXSlL3pKO3DvJIU6Jv9/s1600/novocyte_autosampler.jpg" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="272" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEieTZXtVGMcm3-UKO8Ut_1-aZPg23TBOOm8dBpa-uu17WWPxJgcHHoQpzvaimBUN3ZyuP2G69KTk01ojmCnFvd6sxAd-l5rluuPFeiCq1oE-yCy75SgtFAZwGLTfYXSlL3pKO3DvJIU6Jv9/s320/novocyte_autosampler.jpg" width="320" /></a><span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">The NovoCyte’s fluidics is driven by a peristaltic pump on the sheath side of things and a syringe pump on the sample side of things. They obviously got the memo about the need for a pulseless peristaltic pumps since I wasn't able to detect any fluctuations in sample flow during long runs (observing bead intensity vs. time). Also, the syringe allows for volumetric sample delivery and beadless absolute counts. The fluids are stored in a small tray that sits next to the instrument and holds sheath, waste, and a couple bottles of cleaning solutions used during shutdown. The amount of sheath used is relatively small, and although the tank only holds a liter or two, you can easily eek out a full day's worth of sample acquisition using a reasonable amount of between sample washing. Like any syringe based system, the fluidics seem a bit slow; not in terms of volume per unit time, but simply in the operations that need to take place before and after a sample is acquired. The syringe needs to fill, then push its contents though the flow cell, and if you stop prematurely and have extra sample in the syringe, it needs to expel any extra fluid. Syringe pumps also limit how much fluid you can sample at a time. Whereas positive pressure systems and peristaltic pump systems can acquire freely as long as there's fluid in the tube (and beyond!) syringe-based systems are limited in how much volume they can pull. The NovoCyte pulls between 10 and 100uL of volume at a time, likely ok for many applications, but for those who require a larger sample volume, concatenation is your friend.</span></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">I would argue the heart of the NovoCyte system is its autosampler, and although it’s not a standard feature of the instrument it’s certainly a must-have add-on in my book. It comes with a 24 tube rack that holds 12x75mm “FACS” tubes as well as a microtiter plate holder. The plate holder can accommodate standard 96 well plates (v, flat, and u) as well as 96 deep-well plates and, my personal favorite, 96-tube rack (1.2mL “bullet” tubes racked in the standard 96 well format). Once you’ve mapped out which wells/tubes you’ll be sampling from, the system affords you true, walk-a-way operation complete with email notifications. Sample flow rate, number of washes and mixing is all programmable to allow you some control over optimizing against carryover or for speed.</span></div>
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<div dir="ltr" style="line-height: 1.38; margin-bottom: 0pt; margin-top: 0pt;">
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">As mentioned above, another key usability feature is the system’s one-button startup/shutdown routine. One thing that I like to test on all my instruments is what I call the “cold-start to dots on the plot” time. As the name implies, I’m interested in knowing how long it takes to start the instrument, perform any required start-up routines, run the QA procedure, put on a sample and see dots appear on a plot. For some instruments this can be quite long, sometimes as much as 20-30 minutes. The NovoCyte is extremely fast in this regard. By having a physical button that powers on and performs the fluidics startup, you can do that concurrently with things like logging into the computer, launching the software and preparing for QC. The QC process is bare bones, but actually mimics our standard procedure used on the rest of our instruments - a single peak bead’s MFI and CV are tracked over time and plotted on a L-J plot.</span></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: large; font-style: normal; font-variant: normal; vertical-align: baseline; white-space: pre-wrap;"><u>Optics</u></span></h3>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">The system has 3 Coherent lasers (405, 488, 640) and 13 fluorescence channels as well as the standard 488 light scatter parameters, FS and SS. However, to save cost and space, only 8 detectors are present. Detectors with a standard filter are re-used across multiple lasers. For example, the far red detector that has a 780LP filter detects QD800 when the event passes through the 405nm laser, PECy7 when passing through the blue laser and APCCy7 when passing through the red laser. In theory, this is no different than having those 3 detectors with the same filter on your instrument in physically separate places. In fact, if you opened up any cytometer chances are you would see the same filter repeated on different detectors. By adding in a delay on the detection side of things, the signals terminating at the same detector can be differentiated with just as much accuracy as three separate detectors. Of course, you can test this empirically by running PECy7 and APCCy7 single colors on the NovoCyte and another instrument that has the same filters but on separate detectors and then compare the spillover values. </span></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">When I set up my multicolor panels, I like to stagger the emission across different laser lines. For example, maybe I’ll use BV705, PECy7, and APC together instead of QD800, PECy7, and APCCy7. Doing this on the NovoCyte is a bit more challenging and stacking up 8, 9, or 10-colors in a single panel could pose more spillover issues with this shared detector setup when compared to a 1:1 setup with slightly optimized filters on each detector. It’s for this reason that I don’t think this instrument will actually be a 13-color instrument, but will do just fine with 6 or maybe 8 colors simultaneously. Maxing out the NovoCyte causes an over-population of a few areas of the spectrum, especially the far reds. The table below shows which detectors are shared across lasers. Another point to note is that since the physical detector is shared, that means the "optimized" voltage is shared as well. Whereas normally you might optimize the voltage for BV650 differently than APC, here you're given a single voltage that is "optimized" for both. Let's follow this example a bit further. When I put on unstained cells and adjust voltages, I might expect there to be a higher voltage needed for longer laser/collection filter wavelengths (i.e. more voltage needed for red laser excitation and emission compared to blue or violet). However, with the NovoCyte, I'm given one, predetermined voltage that should work for both. And, to be honest, it does seem to work, although the obsessive compulsive perfectionist inside really wants to fine tune voltages for these two colors independently...but I'll get over it.</span><br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhLRPxaMlhyPUN8UaURhr5t1rgRiUjoNaPgic-LwY6P9FIfE7yk368Gx54JBfOOqooSaV8DsF2biu9__Es0Pwixeu26hFFzpquAnhyphenhyphen6xUoZzXWRHE-b_YVoyLFi0MiSccpxg2rPH8jNuvZi/s1600/NovoCyte_Optical_Bench.jpg" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="195" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhLRPxaMlhyPUN8UaURhr5t1rgRiUjoNaPgic-LwY6P9FIfE7yk368Gx54JBfOOqooSaV8DsF2biu9__Es0Pwixeu26hFFzpquAnhyphenhyphen6xUoZzXWRHE-b_YVoyLFi0MiSccpxg2rPH8jNuvZi/s320/NovoCyte_Optical_Bench.jpg" width="320" /></a></div>
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><br /></span></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: large; font-style: normal; font-variant: normal; vertical-align: baseline; white-space: pre-wrap;"><u>Electronics</u></span></h3>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Similar to the BD Accuri C6, Acea Biosciences chose to go with a fixed voltage system to allow for ease-of-use. It’s well known that pretty much the only thing an end-user can screw up when acquiring data is improperly adjusting the parameter voltages. Having the voltage too high or too low can have dramatic effects on the data. It’s also safe to say that if you looked at the voltages on your cytometer across many people's applications you’d likely find a small range of paramater voltages. So, it’s not surprising that setting a fixed voltage can definitely be beneficial to the masses. Of course there will always be those exceptional cases where you have super bright staining or highly autofluorescent cells, but again that’s the exception, not the norm. As you’d typically find, the spacious 7-log scales, when used with real samples, shrinks down to about 4 decades as the unstained populations typically fall around 10^3. However, the fixed voltage, easy setup philosophy pays dividends when you apply it to a standard workflow. For example, I can walk into the lab, click the startup button, run QC, load a plate, setup my well collection criteria (# of cells, volume, time) and click go. I don’t have to open a bunch of plot and setup voltages or anything like that. I could conceivably collect data blind, dump it into FlowJo and be done. Workflows don’t get much easier than that. Anticipating some complaints in this regard, Acea Biosciences allows for admin-level adjustment of the voltages. This could be handy for those edge case situations where you need a bit more dynamic range, however, as noted above, you still need to be careful since you're actually adjust the voltage for as many as 3 parameters, not just one. </span></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: large; font-style: normal; font-variant: normal; vertical-align: baseline; white-space: pre-wrap;"><b><u>Software</u></b></span></h3>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">I was a bit perplexed at first when it came to using the software. The sample tube navigation area (akin to DiVa’s browser window) is quite DiVa-like. There’s a top-level container (experiment) with a sub-level group (specimen) that contains samples (tubes). It also allows you to differentiate between group level settings and analysis and tube level settings and analysis. Here, instead of copying and pasting between tubes/groups, you can use a FlowJo-esque drag and drop functionality. The software operates in two modes, and you can switch modes on the fly, that is, you can go from a live acquisition view to a data analysis view. As I’ve mentioned before, I don’t care too much for data analysis features clogging up my acquisition software. One of my key complaints against spending resources developing analysis tools is that anything you build into the acquisition software is not going to be as fully-featured as FlowJo, so why bother. I like to use the example that there are no acquisition software packages that do cell cycle modeling. Well, I can’t use that argument anymore since the NovoCyte software does in fact have cell cycle modeling built-in. I don’t think this changes my view, but if you were in a lab that had one instrument and no prior analysis support, this could be a case where you might acquire and analyze in one place. Outside of that, the software can be as uncluttered as you'd like. Again, since there's no need to adjust voltages, there's really no need to sift through an endless sea of plots and region hierarchies. </span></div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhL2jpDopGHNdImU4XGZvqA0bUFVr7fLH2IrMChotn2UmRhQQp7p4c4AhLdM3WYV4psHLu1M10AgoZNqRydVOz5xacQIkp3BfhsTJnozka777mJZg0KJf8HktsdI4PV5HRq19tXKAyf63tg/s1600/NovoCyte_8PeakFigure.jpg" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhL2jpDopGHNdImU4XGZvqA0bUFVr7fLH2IrMChotn2UmRhQQp7p4c4AhLdM3WYV4psHLu1M10AgoZNqRydVOz5xacQIkp3BfhsTJnozka777mJZg0KJf8HktsdI4PV5HRq19tXKAyf63tg/s320/NovoCyte_8PeakFigure.jpg" width="227" /></a></div>
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">Using the Spherotech 8-peak Ultra Rainbow bead set, you can get a glimpse of both the resolution as well as the range among all the channels. Here, they are separated by Laser line (colored bar along the left) as well as which detector is being used. Remember, the plots colored red (for example) are all representing the same detector and same filter, just time gated according to the laser intercept. The differences in resolution, therefore are not a function of the detector or filters, but simply a difference in excitation wavelength and power, which are exciting the mixture of impregnated dyes differently. </span></div>
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<span style="font-family: Arial;"><span style="font-size: 14.6667px; line-height: 20.24px; white-space: pre-wrap;">Dim population resolution is sort of a mixed bag. While the system performs well using the Blue Laser (FITC, PE, PECy7), APC and Pacific Blue are on the high end of resolution. Although the data is not displayed below, other areas where resolution didn't look as good were the red emission channels off the violet laser line (QD800, or BV650, for example). Don't get me wrong, they're still usable, you just won't be using them for your low abundance antigen. </span></span><br />
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<span style="background-color: transparent; color: black; font-family: Arial; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><b>Linearity</b></span></h4>
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<b style="font-weight: normal;">End-to-end linearity was good. Looking at stained CENs, you can see a slight deviation at the higher end of the channels, when normalized against the 2N population. The residuals from the perfect 1:1 line only deviate a little over 3%, which is quite good. The other interesting thing is the fact that it is quite easy to fit many generation of nuclei on a single scale due to the dynamic range on the NovoCyte. Here we are calculating values from 1 to 12 nuclei with room to spare!</b><br />
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<b style="font-weight: normal;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiVp5BLmN5In-XcNk0OYF6wHFUobeTIXe28mH_3Ijf113Y2mtR3zWJ6Q7lhuk-88JAAOA2P-H7ps2J08DHjM-qoGt-QRVL_bW36yZCXHySpSiUm78nnFimxDOQdgs_rOvOcLRs2a1QNkmIg/s1600/NovoCyte_Linearity.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="217" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiVp5BLmN5In-XcNk0OYF6wHFUobeTIXe28mH_3Ijf113Y2mtR3zWJ6Q7lhuk-88JAAOA2P-H7ps2J08DHjM-qoGt-QRVL_bW36yZCXHySpSiUm78nnFimxDOQdgs_rOvOcLRs2a1QNkmIg/s320/NovoCyte_Linearity.png" width="320" /></a></b></div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-style: normal; font-variant: normal; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;"><b>Carryover</b></span></h4>
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For the carryover test, I used the default wash options for the NovoCyte sampler. Here I used the same PI stained CENs from the linearity test, but alternated wells with and without PI. In well 1, I ran PI stained CENs (not shown above). In well 2, I ran unstained CENs (first histogram overlay). In well 3, I ran PI stained CENs (second histogram overlay), in well 4 I ran unstained CENs after 1 wash (3rd histogram overlay), and in well 5, I ran unstained CENs after a 2nd wash (4th histogram overlay panel). What you can see, is in the 3rd histogram overlay, immediately after running PI stained CENs, you can see both cellular carryover (the red peak composed of 372 events compared to the background well of 100 events) as well as dye carryover (shown by the increase in MFI of the unstained CEN peak - blue histogram MFI). After the 2nd wash and collection of unstained CENs (4th histogram overlay) the carryover count goes back down to background levels and the MFI of the PI- peak (blue histogram) goes back down to background levels (more or less). So, to achieve less carryover, one would simply do a 2x wash between wells instead of a 1x wash. </div>
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<span style="background-color: transparent; color: black; font-family: Arial; font-size: large; font-style: normal; font-variant: normal; vertical-align: baseline; white-space: pre-wrap;"><b><u>Wrap-up</u></b></span></h3>
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<div dir="ltr" style="line-height: 1.38; margin-bottom: 0pt; margin-top: 0pt;">
<span style="background-color: transparent; color: black; font-family: Arial; font-size: 14.666666666666666px; font-style: normal; font-variant: normal; font-weight: 400; text-decoration: none; vertical-align: baseline; white-space: pre-wrap;">I think the killer feature of the NovoCyte is usability. It performs well enough on all fronts, and although its specs may not be the very best, I think it more than makes up for that with its ease-of-use. Don’t expect it to blow you away by its performance, but then again, it does a fine job. The other variables that come into play when working with a new company is support and service in the field. Will they have enough staff to support instruments located all over the U.S. or possibly worldwide? This is somewhat of an unproven issue. Instrument owners in the field have been pleased for the most part, and one thing that repeatedly comes up is the responsiveness of Acea Biosciences with regards to issues or feature requests. I guess that's one good thing about working with a startup company, they can't afford to lose any customers or get any bad press. Again, aside from its above average usability feature, I think what sets the NovoCyte apart from its competitors will also be price. For the right price, this could very well be the best instrument around...depending on its price. </span></div>
Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com3tag:blogger.com,1999:blog-8037707352330428788.post-91243220059137525142015-03-26T13:12:00.001-05:002015-07-20T12:39:42.283-05:00Index Sorting - From FACSDiVa to FlowJo<span style="font-family: inherit;">We recently upgraded our FACSAria to FACSDiVa 8 running on Windows 7 primarily for the ability to do index sorting. Getting used to a brand new set of DiVa issues and quirks has been difficult, but we soldiered on nonetheless. After scouring the web for resources on both index sorting and analyzing index sorting data outside of FACSDiVa, I decided to compile all the resources in one place. They are out there, it's just a pain to jump around to various sites trying to compile all the information together. I've done the leg work already, so read on to get the info. Of course, I'm sure there are more elegant ways of doing this in other programs or even in FlowJo, but I needed this info yesterday, so I'm documenting it here for future reference. </span><br />
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<span style="font-family: inherit;"><b>Figuring out index sorting in FACSDiVa.</b> You may think index sorting is no more than checking a box in FACSDiVa, but there are enough one-off situations that arise that it really warrants a separate FAQ. There are two resources that are quite helpful in figuring this part out. The first, oddly enough, is BD's very own <a href="https://www.dropbox.com/s/as02cw1eyow7xop/23-11221-00_Aria_add.pdf?dl=0" target="_blank">Index Sorting Manual</a> (<-- fixed bad link), which comes as an addendum to the FACSDiVa software manual and may not even be installed on your computer or available for download from BD's website. I only came upon this after our BD service engineer sent me a copy of it. The second resource is a document presented at GLIIFCA 2014 by Matt Cochran (University of Rochester), in which he outlines some of <a href="https://www.dropbox.com/s/0wvaxtkvkeoil5f/Cochran_Using%20Index%20Sorting.pdf?dl=0" target="_blank">his tips and tricks</a> for working with index sorting in FACSDiVa 8. </span><br />
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<span style="font-family: inherit;">So, let's assume you figure out how to successfully perform an index sort in FACSDiVa. You should have a Pre-sort FCS file of your entire population, and an Index sort "tube" for each plate you ran. You can export both (or all) of these as FCS files. There is a decent interface for looking at your index sort plate information within FACSDiVa, but if you're use to doing all your analysis is FlowJo, you probably want to bring that data over at some point. And here's the fun part.</span><br />
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<span style="font-family: inherit;"><b>Analyzing index sorting data in FlowJo.</b> I have an application where a user is index sorting based on a range of FITC intensities. The resulting plate will be a mix of FITC low and FITC high clones. The goal of index sorting, in this case, is to retain the original FITC intensity information for each well after the sort. What follows below is <b>A</b> method (not <b>THE</b> method) I stumbled upon to go from an index sort file from FACSDiVa to Figure 1 below. I'd really love for someone to tell me there is a way easier way to do this in FlowJo.</span><br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjUzQYNfHnwhBFpnBGzfuQxfIlsaG1dHgq0lENjrlXBetKgJm4kpnW0ZG6OiQZ0qV8w6eltwgjozMT_rcVTbo97vbkFcC8e46F4JcWMBv6G1uFZbW1WZEJrssmF8zdTkWavsx_3ug63c_sJ/s1600/Plate1_IndexSort.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><span style="font-family: inherit;"><img border="0" height="249" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjUzQYNfHnwhBFpnBGzfuQxfIlsaG1dHgq0lENjrlXBetKgJm4kpnW0ZG6OiQZ0qV8w6eltwgjozMT_rcVTbo97vbkFcC8e46F4JcWMBv6G1uFZbW1WZEJrssmF8zdTkWavsx_3ug63c_sJ/s1600/Plate1_IndexSort.jpg" width="320" /></span></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: inherit; font-size: x-small;">Figure 1. Heatmap analysis of index sorting file.</span></td></tr>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj4FCrWoOzJUR3qbyPmefWxpKTidPe0iEDgoE1osZxajtwsisgTTkUeXj9lFK8Nx4y6D9UhE5ubzPbsONzck3U-5-RE9hJ6numfT8y-pdBFrkJtHPSUiq6z9mYHEF-tnqgdJQB7iNuwl_UZ/s1600/RunScript.tiff" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><span style="font-family: inherit;"><img border="0" height="207" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj4FCrWoOzJUR3qbyPmefWxpKTidPe0iEDgoE1osZxajtwsisgTTkUeXj9lFK8Nx4y6D9UhE5ubzPbsONzck3U-5-RE9hJ6numfT8y-pdBFrkJtHPSUiq6z9mYHEF-tnqgdJQB7iNuwl_UZ/s1600/RunScript.tiff" width="320" /></span></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><span style="font-family: inherit; font-size: x-small;">Figure 2. Running the initial script to create 96 populations</span></td></tr>
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<span style="font-family: inherit;"><b>Step 1</b>: Use the <a href="http://flowjo.typepad.com/the_daily_dongle/2015/01/script-editor-keyword-and-index-sorting-example.html" target="_blank">Script Editor index sorting example</a> from the Daily Dongle Blog (or see Addendum below regarding the method in Version 9). You simply copy and paste the script starting with <i> </i><span style="background-color: white; line-height: 20.2159996032715px;"><i>/** --- Iterate samples --- **/</i> all the way through </span><span style="background-color: white; font-style: italic; line-height: 20.2159996032715px;">gate.update(); </span><span style="background-color: white; line-height: 20.2159996032715px;"><i>}</i> and paste it into the script editor window (under the tools tab of the ribbon) in version 10.0.7 (if you have access to the 10.0.8beta version, I would do this step in that version...you'll see why later). Highlight your index sorting file in the workspace and click the run button in the script editor window. You should now have 96 populations under your index sorting file. If you end up with a bunch of "-" where it usually says the number of cells in each population, click the refresh button at the top of the workspace window and then it'll show you that there is 1 cell per region (Figure 2). </span></span><br />
<span style="font-family: inherit;"><span style="line-height: 20.2159996032715px;"></span><br /><span style="line-height: 20.2159996032715px;"></span><span style="background-color: white; line-height: 20.2159996032715px;"></span></span>
<span style="background-color: white; line-height: 20.2159996032715px;"><span style="font-family: inherit;"><b>Step 2:</b> The next step is to export each of these populations as its own FCS file. In essence creating 96 FCS files. The problem here is that you can do the initial index sorting script in version 10.0.7, but you can't do the export to 96 FCS files in 10.0.7 for the Mac (I think you can do this in the windows version, but I'm not sure). You can do the export to 96 separate files in Mac version 9.8.3, but you can't do the initial script in 9.8.3. So, if you can do this all in 10.0.8beta, that's your best bet (or on windows). So, in 10.0.8b, you can highlight all the populations and choose export (right click or within the File tab in the ribbon) and export this as 96 FCS files. </span></span><br />
<span style="background-color: white; line-height: 20.2159996032715px;"><span style="font-family: inherit;"><br /></span></span>
<span style="background-color: white;"><span style="font-family: inherit;"><b style="line-height: 20.2159996032715px;">Step 3:</b><span style="line-height: 20.2159996032715px;"> </span><b style="line-height: 20.2159996032715px;">Using the plate layout to create a heatmap.</b><span style="line-height: 20.2159996032715px;"> The last step is to load the 96 FCS files into FlowJo v10.x.x and assign the Well ID keyword to each of the files corresponding to their position on the plate. Now, the files are in chronological order going across and then down (in serpentine fashion). So all you have to do is add the Well ID keyword as a column and copy and paste a list of Well IDs (A1 - H12 in serpentine fashion) from a spreadsheet. BUT WAIT, THERE's MORE! If you're doing this on a Mac, </span><a href="http://flowjo.typepad.com/the_daily_dongle/2015/02/copy-keyword-values-from-excel-to-flowjo-x-mac.html" style="line-height: 20.2159996032715px;" target="_blank">this post from the Daily Dongle</a><span style="line-height: 20.2159996032715px;"> states that since Mac Excel copies data in the ANSI format you won't be able to paste into FlowJo, which only reads the Unicode format. To get around this, create the Well ID list in Google Sheets and copy and paste from there (Google Sheets copies data in Unicode format). Now that you have a Well ID associated with each of the files you can <a href="http://docs.flowjo.com/vx/experiment-based-platforms/plate-editor/visualizing-plate-based-data/" target="_blank">use this link to in FlowJo's documentation</a> to set up a heatmap of your index sorting data.</span></span></span><br />
<span style="background-color: white;"><span style="line-height: 20.2159996032715px;"><span style="font-family: inherit;"><br /></span></span></span>
<span style="background-color: white;"><span style="line-height: 20.2159996032715px;"><span style="font-family: inherit;">And there you have it. Please leave a comment below with your preferred method of analyzing index sorting data using whatever software you like. </span></span></span><br />
<span style="background-color: white;"><span style="line-height: 20.2159996032715px;"><span style="font-family: inherit;"><br /></span></span></span>
<span style="background-color: white;"><span style="line-height: 20.2159996032715px;"><span style="font-family: inherit;"><b>Addendum #1</b>: Using the script is somewhat cumbersome. Thanks to Helene Dujardin (from HCD Bioexperts) for the tip below:</span></span></span><br />
<span style="background-color: white;"><span style="line-height: 20.2159996032715px;"><span style="font-family: inherit;"><br /></span></span></span>
<span style="background-color: white;"><span style="line-height: 20.2159996032715px;"><span style="font-family: inherit;">"</span></span></span><span style="background-color: white; color: #404040; font-family: Roboto, arial, sans-serif; font-size: 13px; line-height: 18.2000007629395px;">There is another way in version 9, has there is an option for index sort analysis. Select your sample and go to the menu Platform/ Event number gate / Create Indexed sort gates. It will directly create a gate for each of your well. Each gate name will be the corresponding well ID.</span><br />
<span style="background-color: white;"><br style="color: #404040; font-family: Roboto, arial, sans-serif; font-size: 13px; line-height: 18.2000007629395px;" /><span style="color: #404040; font-family: Roboto, arial, sans-serif; font-size: 13px; line-height: 18.2000007629395px;">You can then export each of your gate as a new fcs file also with version 9. Your exported fcs file name will include the well ID if your original fcs file name is not too long (you can change it by changing the $FIL keyword)."</span></span><span style="background-color: white; font-family: Helvetica, Arial, sans-serif; font-size: 14.4399995803833px; line-height: 20.2159996032715px;"><br /></span><br />
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<span style="background-color: white;"><span style="color: #404040; font-family: Roboto, arial, sans-serif; font-size: 13px; line-height: 18.2000007629395px;"><b>Addendum #2</b>: Using the methods outlined in Addendum #1, I'll add one more point of interest. When you export the Index FCS file from FACSDiVa, you might get a really long name (Specimen_001_Index_Tube_001.fcs). FlowJo v9 freezes when you try and export all the regions as FCS files, so you'll need to rename the files after you import the parent into FlowJo. I've been renaming them (CMD+R shortcut) INDX_1, INDX2, etc... Now, when you export the regions as FCS files they'll be labeled INDX_1_A01, etc...</span></span>Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com5tag:blogger.com,1999:blog-8037707352330428788.post-53291621350339203712014-12-31T17:10:00.001-06:002014-12-31T17:11:50.769-06:00Flow Cytometry Core Facility New Year's Resolutions<div class="separator" style="clear: both; text-align: center;">
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It's that time of year again when the gyms are packed and weight-loss commercials air continuously.This year, why not turn you attention towards your core facility and come up with some resolutions the whole lab can take part in. The best part is you'll have help from the rest of your lab mates to keep you on task.<br />
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So, just as we do with our personal lives, allow me to present an ambitious 10 resolutions for the UCFlow core facility. Presented in no particular order, I give you:<br />
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<ol>
<li>I'd love to devote more time towards taking better care of our instruments, in terms of routine maintenance and a more streamlined QA process across the board.</li>
<li>Do a better job getting administrative tasks like billing/invoicing/usage tracking/usage analysis done on-time and with greater regularity.</li>
<li>It's always nice to see how the work done in the core fits into the bigger picture, so I would like to go to more of my user's talks on campus.</li>
<li>It's pretty clear data analysis is a hot topic these days, so I want to focus more attention on <a href="http://youtu.be/_B7mo6dB3BU" target="_blank">complex data analysis solutions for users</a> (is R worth it?, try more advanced stuff in Cytobank or FlowJo?, etc...)</li>
<li>Who can't use more/new instruments. You'll get none of the instruments you don't write a grant for. I think I need to be more aggressive in my pursuit of new funding sources for instrumentation.</li>
<li><a href="http://ucflow.blogspot.com/" target="_blank">Blog more often</a> (a perennial resolution for me).</li>
<li>I'm convinced that the Hangouts on Air that we do in the <a href="https://plus.google.com/communities/107840035426159582772" target="_blank">Cytometry Community on Google+</a> are super useful, and so I'd like to turn that into a more regular thing. </li>
<li>I've always thought that eventually core facilities would collapse into each other to create mega technology centers. But, before that happens, I would like to start by increasing interactions with other core facilities on campus to see what they're doing and what's new in technology in other fields.</li>
<li>It use to be the rule in our core that if you went to a meeting, you had to present something. I haven't been as faithful to that rule as I would like, so I'm bringing it back.</li>
<li>Of course this last one happens all the time, but I would like to focus some attention on re-evaluating facility costs with greater scrutiny to determine where reductions can be made.</li>
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Well, there you have it. I just hope I'll be able to hold onto these longer than my annual attempts to get back "in shape." How about you? Any resolutions you'd like to add for your core facility? Leave a comment.</div>
Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com0tag:blogger.com,1999:blog-8037707352330428788.post-38266987351149502062014-12-19T13:16:00.000-06:002014-12-19T13:16:16.272-06:00Core Facility Acknowledgment Accounting 101 - How to make sure your work is being recognized.<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="http://www.biotechniques.com/BiotechniquesJournal/2014/December/From-the-Editor/biotechniques-355619.html" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;" target="_blank"><img alt="http://www.biotechniques.com/BiotechniquesJournal/2014/December/From-the-Editor/biotechniques-355619.html" border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgE0SepZJcaX_xxoXHCKU9QLOxMfEc603cpUdUCxMKsLKJ2II1VpuogJAhN5Asy-ktbtDR2ZDfFPJfy1PLz1xnbDf8Tuxvv8PwK_d2zvMRIv5GP53X_fUoLHlc2mu2_fd0pFJjP9p3WLHz1/s1600/biotechniquesarticle.tiff" height="320" width="243" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">BioTechniques Article on SRL Attribution</td></tr>
</tbody></table>
A <a href="http://www.biotechniques.com/BiotechniquesJournal/2014/December/From-the-Editor/biotechniques-355619.html" target="_blank">recent article in Biotechniques</a> has spurred some interesting discussions in the Academic Core Facility (or as we cytometry cores like to call them, Shared Resource Laboratories - SRLs) world. The gist of the article states that all too often core facilities are not properly acknowledged in publications that clearly are using the services provided by their institutional cores. The flip-side of this argument is that investigators are already paying for the services rendered so that fee is essentially all the "acknowledgment" that is required. However, since many times core facilities are partially funded by government agencies, the services (and more accurately the service recharge rates) are being subsidized. Therefore, the <i>payment</i> isn't <i>payment</i> enough.<br />
<br />
Whether you agree or disagree with this basic tenet is really beyond the scope of this post. What I'd like to share here is my way of fostering the proper relationship with my users such that they feel compelled to acknowledge the excellent work of the core instead of feeling obligated to do so. <br />
<br />
What follows is basically a three-part approach to accomplishing the goal of being acknowledged as a core facility in publications that utilize your services. The reason you may wish to do this could vary, but likely involve justification of your core facility's existence to your institution's administrators or various "Centers" you may receive funding from. For example, as part of the <a href="http://cancer.uchicago.edu/" target="_blank">University of Chicago's designation as a Comprehensive Cancer Center</a> from the NCI, we must keep track of cancer-related publications that utilize our core facility. So, obviously it would be easiest for us to search PubMed for the inclusion of our core facility's name or even the cancer center support grant number in the reference. However, many times our facility is omitted from the acknowledgement section of the publication. To help modify this behavior, we need to first find the publications, then organize them, and lastly reach out to our authors/users to help them understand why acknowledgements are important. Here are these steps.<br />
<br />
<b>Part 1 - Finding publications that should designate attributed to your core facility's work.</b><br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiwQcqqUrLyC34p7GAquW9tLj7MYN_uL3dOfBswGuenH5Z2QtH_J_XlxmFFHaH9ju7POEQBnEtUCoH2ttswG3XQedEIdN3yn-KJ7JA51MimSEKijhtxUO_xsHHqkvbAIYTxhTu5iDYHN0hi/s1600/keywordsforUCFlow.tiff" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiwQcqqUrLyC34p7GAquW9tLj7MYN_uL3dOfBswGuenH5Z2QtH_J_XlxmFFHaH9ju7POEQBnEtUCoH2ttswG3XQedEIdN3yn-KJ7JA51MimSEKijhtxUO_xsHHqkvbAIYTxhTu5iDYHN0hi/s1600/keywordsforUCFlow.tiff" height="167" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Fig. 1 - Keywords to find references based on your core's services.</td></tr>
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You can use the "Saved Searches" functionality within PubMed to find relevant articles and have them emailed to you directly as soon as something meets the search criteria. There's already a good <a href="http://www.nlm.nih.gov/bsd/disted/pubmedtutorial/040_015.html" target="_blank">tutorial on PubMed</a> that will walk you through the steps, so I won't go into that in great detail, but let me summarize my steps.<br />
<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgt0JIn123-WEP_OWTr2f1A1MI0IicZrbIof58IyJHu9Rm4k-YHz_aBW6WLZlXwzxE8x2HpmP_fsASPPRiXCzm3998_3xJXtiwpQJdg4iIBYI_MJmn72wj9HutrzSEV3I1Om_BqnlWsc-HF/s1600/history_1.tiff" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgt0JIn123-WEP_OWTr2f1A1MI0IicZrbIof58IyJHu9Rm4k-YHz_aBW6WLZlXwzxE8x2HpmP_fsASPPRiXCzm3998_3xJXtiwpQJdg4iIBYI_MJmn72wj9HutrzSEV3I1Om_BqnlWsc-HF/s1600/history_1.tiff" height="135" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 2. Part #1 of search yields over 168,000 results.</td></tr>
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I jump right into the <a href="http://www.ncbi.nlm.nih.gov/pubmed/advanced" target="_blank">Advanced Search Builder</a> in PubMed using various keywords for different parts of the search structure. For example, I limit the search results to an affiliation of University of Chicago. There are a few external users that I'd like to track as well, but I put them in a separate search. The first part is to put in keywords based on any part of the text that your users may use to describe what they did in your facility. Remember, many users refer to any part of flow cytometry as "FACS" so you'll want to make that part of your search criteria. Figure 1 shows you some of the ones I use (note the use of 'Or' boolean to search on any of these keywords). <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjPegrC10lvCFrYEvCE1i5V85zNaQBqIMmIURI7C9gHFcbmFgN0IPExrBZnKC8DmdKgT1uy_MURtLMpK6OaJwBiDh_xgRlEmzz3xjSb-gc5NglePZUVtWTvJeCfuWL72w3Cx0AJELshyphenhyphenPB1/s1600/builder_plus_affiliation.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjPegrC10lvCFrYEvCE1i5V85zNaQBqIMmIURI7C9gHFcbmFgN0IPExrBZnKC8DmdKgT1uy_MURtLMpK6OaJwBiDh_xgRlEmzz3xjSb-gc5NglePZUVtWTvJeCfuWL72w3Cx0AJELshyphenhyphenPB1/s1600/builder_plus_affiliation.tiff" height="149" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 3. Search restricted to affiliation of University of Chicago</td></tr>
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<br />
Next I use the "Add to history" link near the search to hold onto those search results temporarily (Figure 2).<br />
<br />
I click the "Add" link next to search #1 to add these 168,000+ results back into the builder, and then refine the search by using the "And" boolean and restricting the "Affiliation" field with 'University of Chicago (Figure 3.)<br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="clear: right; float: right; margin-bottom: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgXK-He3kgkl4GIEBxqSX8tL6KFTtp79c2-3p87fnZd926nWrasdqpACddTdQcc8WYiKivNjg56kg-uMliM40UCVLT5aS2ZKtjGxCmhbCoB9BvVyNgvdMUyPkAGbFu-mPmbHGNKPRmxEXE2/s1600/searchresults.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgXK-He3kgkl4GIEBxqSX8tL6KFTtp79c2-3p87fnZd926nWrasdqpACddTdQcc8WYiKivNjg56kg-uMliM40UCVLT5aS2ZKtjGxCmhbCoB9BvVyNgvdMUyPkAGbFu-mPmbHGNKPRmxEXE2/s1600/searchresults.tiff" height="250" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 4. Search based on keywords, affiliation, date range</td></tr>
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You can further refine the search based on Date ranges or excluding reviews or a bunch of other search criteria using the same strategy (Add to history, then add those results back to the Builder and refine again). I find this method of going back and forth between the history table and the builder easier than trying to put everything into one complex boolean structure. Click search to view your results (Figure 4).<br />
<br />
Once you've created your search criteria and confirmed that it is giving you what you've intended, you'll want to save the search, using the "Save search" link below the search box. Figure 5 shows you some of the options available for setting up the saved search. Note that you'll need a PubMed profile to set this up, so the first time you try and save a search, it'll ask you to create an account. Here, I've chosen to send me an email<br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi5ca1ObrStyPUTXuir4-i8QK1ybqZTPqvHiZDkzy1-Od49DgxgORcRlCm6KkmOjs0J8CSLKbVTa2-k024fA947FhVnd7bEmF3Fyf6yH3lrt1PsIXR40IUIgQEZ3QR1EZSphI5pzASYAq3u/s1600/savesearchemailtome.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi5ca1ObrStyPUTXuir4-i8QK1ybqZTPqvHiZDkzy1-Od49DgxgORcRlCm6KkmOjs0J8CSLKbVTa2-k024fA947FhVnd7bEmF3Fyf6yH3lrt1PsIXR40IUIgQEZ3QR1EZSphI5pzASYAq3u/s1600/savesearchemailtome.tiff" height="320" width="257" /></a></td></tr>
<tr><td class="tr-caption" style="font-size: 12.8000001907349px; text-align: center;">Figure 5. Saving the search and setting up email digest.</td></tr>
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weekly on Mondays (when I'm likely to have free time) so I can review the new references. I've also placed some text (or even a custom #) so that I can filter my email properly and it doesn't get lost amongst the email clutter. I save the search and wait for the emails. By the way, you can now set up all sorts of notification. For example, I've recently been doing a lot of microparticle stuff, so I have a separate digest setup to send me email notifications of new publications using flow/image cytometry to analyze microparticles (or microvesicles or micro particles, etc...)<br />
<br />
<br />
<b>Part 2 - Organize references and tag them to easily create reports later.</b><br />
<br />
In part 2, my goal is to receive these email notifications, skim through the publication and then find a way to organize the references neatly and efficiently.<br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="clear: right; float: right; margin-bottom: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgbIeTGNzpxgA7fB0igiWknnhmPwJNORpkpQZsnS_fPgE5aStD5pqz7Vq8wPkLCTs7d4Z6_O3UP3WUsgvQe5diRiqhqokzfiOyEBvTxorrUVGN7BV_sfUSOCP95hmLMciYMtY2_UrOgEw2d/s1600/EmailedDigest.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgbIeTGNzpxgA7fB0igiWknnhmPwJNORpkpQZsnS_fPgE5aStD5pqz7Vq8wPkLCTs7d4Z6_O3UP3WUsgvQe5diRiqhqokzfiOyEBvTxorrUVGN7BV_sfUSOCP95hmLMciYMtY2_UrOgEw2d/s1600/EmailedDigest.tiff" height="195" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 6. Email notification from My NCBI</td></tr>
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The emails arrive in my inbox on Monday mornings as references become available. If there are no new references, you will not get an email. Figure 6 shows an example of what this email looks like.<br />
<br />
Next, I follow the link, and read through the manuscript to ensure the work being reported was in fact from my core. If I'm unsure, I can always ask the author, but I tend to recognize work done on my instruments.<br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiOarR23i1FVmjNG8TUdl29ne1ROAQPI-PmbqCLEKMaYyhuVMV4f6nQ6PhArFT9nDRLSg40IU0C0sW-iDJgROWIuPGjhf4ffXp8XHXScvx6XAcWXbrYeiJe4CbB1ZKmiJJpWgTVLqfALlUx/s1600/savetozotero.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiOarR23i1FVmjNG8TUdl29ne1ROAQPI-PmbqCLEKMaYyhuVMV4f6nQ6PhArFT9nDRLSg40IU0C0sW-iDJgROWIuPGjhf4ffXp8XHXScvx6XAcWXbrYeiJe4CbB1ZKmiJJpWgTVLqfALlUx/s1600/savetozotero.tiff" height="200" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 7. One-click add to Zotero button in URL bar</td></tr>
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One thing that becomes evident is you need to have a way to manage all these references. There are a ton of ways to do this from the most rudimentary word doc or spreadsheet to sophisticated software management tools. The tool I like for this part is <a href="https://www.zotero.org/" target="_blank">Zotero</a>. It's similar in function to things like <a href="http://endnote.com/" target="_blank">Endnote</a> or <a href="http://www.mendeley.com/" target="_blank">Mendeley</a>, but it's basically an organization tool for references. The part I like most about Zotero is that there's a Chrome extension that allows one-click adding of references to my database (Figure 7). Plus it will go out and find the PDF of the full-text reference and store that locally as well (when available). It lives in the cloud and can be accessed anywhere. <br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgfYrBvApJSdweZKwJgE9Kla1Qv8rywVvKW9Fc3v8B_N2uZXNmMUnI5ye2SOPPXaoFepy7YRtEHKELc2PNT14y6OYtkJ_nplmQqvWilUubQM54STg_QT-n_cpaALmliJRXOHXlSahkHRLQy/s1600/ZoteroInput.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgfYrBvApJSdweZKwJgE9Kla1Qv8rywVvKW9Fc3v8B_N2uZXNmMUnI5ye2SOPPXaoFepy7YRtEHKELc2PNT14y6OYtkJ_nplmQqvWilUubQM54STg_QT-n_cpaALmliJRXOHXlSahkHRLQy/s1600/ZoteroInput.tiff" height="180" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 8. Zotero Organizing tool for references (running on Mac)</td></tr>
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Once in Zotero (Figure 8), I can add tags to the references to help organize them further. I like to tag things by services used (Cell Sorting vs. Analyzer Usage vs. Other things), Instrument referenced (e.g. FACSAria), Whether this could be used for my Cancer Center grant renewal (UCCCC), and other informative tags. Then, down the road when I need to pull up some justification for a new sorter, I can include a list of publications that utilized the cell sorting service or maybe even a specific sorter.<br />
<br />
This makes organizing and searching through references a breeze.<br />
<br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="clear: right; float: right; margin-bottom: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiEdbKAWGnSf_HFkF1yo7ZA59iSSB3-6ldXyEysHpG22uYzQm7zNlT5jen8tSNw9s1VSNQvy6VKTJyjSgB9LVJ69BJBFC3889i4fIrKzw6G6s2ZPm0DNPylh4H9DXB_9bzI5WeASxDaAJHM/s1600/thanksforack.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiEdbKAWGnSf_HFkF1yo7ZA59iSSB3-6ldXyEysHpG22uYzQm7zNlT5jen8tSNw9s1VSNQvy6VKTJyjSgB9LVJ69BJBFC3889i4fIrKzw6G6s2ZPm0DNPylh4H9DXB_9bzI5WeASxDaAJHM/s1600/thanksforack.tiff" height="320" width="274" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 9. Thanks for the acknowledgement</td></tr>
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<b>Part 3 - Compel investigators to acknowledge your core facility.</b><br />
<br />
Now comes the hard part. How to suggest to your facility users that they should be acknowledging your core without sounding like a jerk.<br />
<br />
As I'm skimming references, I'll quickly jump to the acknowledgement section and check for recognition of the core, or perhaps individual members of the core (either is fine with me). If the user does acknowledge the core, I make sure to send them an email thanking them for doing so. This positive reinforcement goes a long way toward ensuring this type of action recurs in the future. I also explain why it's important to us that the core be acknowledged. An example email is shown in Figure 9. Of course congratulating them on a job well done can only help to sweeten the deal.<br />
<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="clear: right; float: right; margin-bottom: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj-EsB0zautEIz6zGCGQY8Lzc1KZni2D6NOb8nYC0cJmDMQIfuK1XyKGWLUHbEO_l8czKm5Psd3AZ48eeR3HK6nTlXdKeNEWa-RzB8qqfCbjhKFwN0VCMjMsoHVP2JJDpXKwqIzs440GjlG/s1600/noack_nexttime.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj-EsB0zautEIz6zGCGQY8Lzc1KZni2D6NOb8nYC0cJmDMQIfuK1XyKGWLUHbEO_l8czKm5Psd3AZ48eeR3HK6nTlXdKeNEWa-RzB8qqfCbjhKFwN0VCMjMsoHVP2JJDpXKwqIzs440GjlG/s1600/noack_nexttime.tiff" height="320" width="275" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Figure 10. Maybe next time...?</td></tr>
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If I see there is no mention of the core in the acknowledgements or methods section, I'll send a similarly positive email, but ask them to consider acknowledging us in the future. I make sure to include some example text of what I would like them to say, as well as send them a link to the example text on our web site (Figure 10).<br />
<br />
Of course, you can save these emails as templates and simply change the name and journal to personalize them.<br />
<br />
The responses I've received from these emails has been tremendous. I think they are both appreciative of the recognition of their work as well as understanding of the needs of the core to be recognized.<br />
<br />
We all understand the need for metrics such as publications and their importance in validating the success of core facilities. However, instead of taking a passive approach and hoping people read your web site asking to be acknowledged, the method proposed here takes a proactive approach that has already increased the desired result.<br />
<br />
PubMed is pretty comprehensive, but there could be other sources for finding work being discussed that should point back to your core facility. Magazine articles, intra-institutional articles or highlights, blog posts, etc... all should be explored and stored. You can use a series of other rss feeds or Google search alerts to help you find this information too. Asking a PI to mention the core facility in an intra-institutional newsletter is certainly within your purview.<br />
<br />
Happy Hunting!<br />
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Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com0tag:blogger.com,1999:blog-8037707352330428788.post-43642930764352983592014-09-04T20:18:00.000-05:002014-09-04T20:18:31.387-05:00A First Look at the Beckman Coulter CytoFLEX - Strong Performance in a Small BoxOver the past few years, we've been inundated with small, inexpensive cytometers with the promise that they can perform as well as the big boys. Up until now, I would have told you not to waste your time... up until now.<br />
<br />
In 2013, an unknown company called <a href="http://www.xitogen.com/en/" target="_blank">Xitogen</a> set up a booth at the annual <a href="http://cytoconference.org/2015/Home.aspx" target="_blank">CYTO conference</a>. Before long, there was a buzz racing through the exhibit floor aisles of a flow cytometer starting at ~US$25,000 (1 laser, 2 colors). The Chinese company, headquartered in the Suzhou Industrial Park, set out to provide an alternative for Chinese researchers to acquire affordable flow cytometry instrumentation without having to deal with overpriced imported hardware from the big players. With U.S. zero install base, and zero user-generated data, CYTO 2013 came and went, and the buzz surrounding Xitogen died out. It was pretty obvious the better known cytometer manufacturers would be taking a look at the company for a possible acquisition, and in April of 2014, Beckman Coulter announced they would <a href="http://bit.ly/1rpLmyG" target="_blank">purchase Xitogen</a> for an undisclosed amount of money. The acquisition was finalized in June 2014. At CYTO2014, Beckman Coulter revealed the re-branded instrument now called CytoFLEX. <br />
<br />
I had the chance to spend about a month with the CytoFLEX and what follows are some of my thoughts about the key features, successes and failures of this instrument.<br />
<h3>
</h3>
<h3>
General Technical Specs:</h3>
<div>
The CytoFLEX came to me as a 3 laser system including a 50mW 488nm laser, a 55mW 640nm <br />
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<tr><td class="tr-caption" style="text-align: center;">Beckman Coulter CytoFLEX Analyzer</td></tr>
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laser, and a 93mW 405nm laser. The system also has 9 fluorescence channels in a 4-3-2 configuration, respectively. In addition, there are 3 light scatter parameters, the typical blue laser scatter yielding forward and side scatter, and an additional side scatter parameter off the 405nm laser. Pulse height and area are collected for all parameters, and a width signal can be selected for any one of the parameters. The fluidics system is controlled through peristaltic pumps for both the sheath and sample lines, and the sample volume flow rates can range from 10ul/min up to 240ul/min with 10, 30, and 60 ul/min presets (referred to as Low, Med, and Hi, respectively). A single tube holder with built-in backflush loads samples into the instrument one-at-a-time, and the hardware is controlled by the bare bones, but highly functional CytExpert acquisition software.<br />
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The system that is suppose to ship some time in October will be configurable with 3 spatially separated lasers (with a 4th coming soon?), with a variety of laser options and colors available. The base configuration should include 3 lasers, and 13-colors in a 5-5-3 config (violet, blue, red, respectively).</div>
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Look and feel:</h3>
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<tr><td class="tr-caption" style="text-align: center;">A look inside the CytoFLEX revealing lots of unused space.</td></tr>
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The instrument itself is quite small, fitting roughly into a 40cm cube, but even the box itself seems to be too big for whats being housed inside. A peak under the hood reveals a ton of unused space (multiwell autosampler, perhaps!!!). Pretty much every component on this instrument looks like a fraction of its counterpart on more common cytometers. However, it's quite clear that every penny possible was pinched in the manufacturing of this instrument. Everything about it screams cheap. That's not necessarily a bad thing per se, but as soon as you start opening up lids and doors and see some of the components inside, it becomes clear how they were able to create a functional instrument at bargain prices. Beckman Coulter has said that part of what they will do to the CytoFLEX is to add some polish to the components without adding cost. A final product and price point has yet to be revealed, but we expect to see it in the wild this fall.</div>
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<h3>
Fluidics:</h3>
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The Sheath and Waste tanks sit beside the instrument and have a single output/input line, respectively. They hold about 5 liters, which should last most of a day with moderate use and reasonable amounts of backflushing. The preferred sheath for this system is some high quality H<span style="font-size: xx-small;">2</span>O (Insert Waterboy reference here). Beckman Coulter will likely sell you a box of water at a premium and call it "Coulter Sheath" but you'll be just fine grabbing some DI from your MilliQ system.</div>
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Again the system moves fluid throughout using a pair of peristaltic pumps. The non-fluid movement of peristaltic pumps tend to make them not ideal for a system that requires stable fluid flow, but in testing the CytoFLEX, I saw no fluctuations in any of the channels over long runs with beads (plotting bead intensity vs. time). This type of instability due to peristaltic pump oscillation had been reported in some iterations of the Accuri C6 when it first came out. In the CytoFLEX, special attention was paid to create a pulseless peristaltic pump, which definitely holds true in my testing.</div>
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Although the sample volume flow rate has a custom setting that allows it to go up to 240ul/min, in <br />
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<tr><td class="tr-caption" style="text-align: center;">Close-up of the sample tube loading arm.</td></tr>
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my tests, I saw dramatic declines in scatter profiles and less obvious, but still present, losses in resolution of fluorescence profiles beyond 100ul/min. I think the 240 setting would be great for cleaning the sample line out, or maybe forcing through a stubborn clog, but not for collecting data. This type of flow rate is pretty much on par with other hydrodynamically focused fluidics system (unlike, for example the Attune that uses acoustic focusing and can easily go up to 1000ul/min with minimal degradation of profiles). Although the 80um wide beam spots may insulate the wide sample core stream from really poor resolution.</div>
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The sample loading stage is a bit funky at this point. The loading stage moves in and out with the smoothness of 20 grit sandpaper sliding across berber carpeting (i.e. not smooth at all). This loading/unloading operation slows down the process just enough to be annoying, but you get use to it after a while. No plate loader (yet), No multi-tube loader (yet).</div>
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<h3>
Optics: </h3>
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The optical system on the CytoFLEX is the biggest departure from any other instrument developed. A lot of the technology is proprietary, and as much information as I was able to deduce I'll share here, but I could be flat wrong on some things, so take what I say with a grain of salt. </div>
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<tr><td class="tr-caption" style="text-align: center;">Custom made Laser modules</td></tr>
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Don't expect to see the familiar Coherent Laser Cubes on this instrument. In fact, these lasers are custom made, in-house in the Chinese facilities (where the entire instrument is manufactured). When you take off the laser compartment cover, you're greeted with non-descript tiny black boxes with a sticker on them telling you which laser it is. Here is where they can save a lot of money. Without being beholden to the Coherent behemouth, they're not locked into Coherent prices. And, since they are making the lasers themselves, they can customize everything about them according to this specific instrument. Worried about the quality? I was too, until I saw the performance. Of course, what I'm not able to test is long-term laser life on these. The stated spec on Xitogen's web site for lifetime is 20,000 hours, but this hasn't been tested in the field, as far as I know. The air launched beams go through the typical steering and shaping optics and terminate at the flow cell in front of one of a 7 "pinholes" on the instrument. Beam sizes and power efflux are restricted to a 5um x 80um gaussian profile courtesy of the beam shaping optic and its large 1.3 NA, which means most of the laser power gets focused to the "pinhole" in a slit (N.B. I say "pinhole" since it's unclear if there are actual pinholes in the traditional sense or some other sort of voodoo magic). This should allow for maximal excitation of fluors and minimal crosstalk between laser lines.</div>
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<tr><td class="tr-caption" style="text-align: center;">A look at the laser path with the covers off and interlock defeated <br />
(Don't try this at home kids!)</td></tr>
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Emitted light is collected by fiber optic bundles which carry the light to the detector blocks. The detector blocks, referred to as Fiber Array Photo Detector Modules or FAPDs is where all the innovation takes place. The first thing you'll notice when looking inside the FAPDs is the small size of the filter sticks. Pulling one out reveals a tiny piece of glass no more than a few millimeters square. However, the rest of the components inside the FAPD are completely foreign to someone who's looked at dichroics, bandpasses, and PMTs his whole cytometry life. The light exiting the fiber passes through a <a href="http://en.wikipedia.org/wiki/Wavelength-division_multiplexing" target="_blank">wavelength division multiplexer</a>, which acts like conventional dichroics to partially <br />
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<tr><td class="tr-caption" style="text-align: center;">Looking into the FAPD with a filter stick removed.</td></tr>
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split the light into distinct ranges, and then the light is further refined by the bandpass filters before hitting the photodiode. Photodiode? Don't you mean PMT? No, you read that right, this system uses Avalanche Photodiodes (APDs). These semiconductor detectors are well-known for their high sensitivity, and silicon based APDs have good quantum efficiency in the visible and near-IR range as well as low noise. If they're so sensitive why haven't they been used before? Good question, and as far as I can tell, the problem has always been the amount of voltage that needs to be applied to achieve high sensitivity and this high voltage causing breakdown of the APD. Somehow, this has been circumvented in the CytoFLEX. The other interesting thing about the detectors is that the response of the APDs across the entire range is absolutely linear. They stand by this fact so much so that if you set up compensation on FITC vs. PE at one set of voltages, and then change the voltages, the system will automatically adjust the compensation values to take into account the new voltage settings. This can only happen if the response is linear from end-to-end and therefore compensation is merely a mathematical equation with voltages as one of the variables.</div>
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<h3>
Electronics:</h3>
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The system uses 16-bit A/D converters and boasts of 7-decades of dynamic range. Normally 16-bits doesn't get you that much range, but by oversampling the pulses at 40MHz, and adding up all the samples, a full 7-log scale can be achieved. However, like most of these large scales, the first decade tends to exhibit poor resolution and is "hidden" by default. So the scale goes from 10^2 up to 10^7. Qualitatively, I will say that I was able to resolve all 8-peaks of the 8-peak rainbow bead set with some room on both sides of peaks 1 and 8 - that doesn't always happen.</div>
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One of the only complaints I had about this instrument was the loss of data at moderate to high event rates. This has to be due to the pulse processing speed of the electronics system and its inability to process the signals fast enough. It's likely influenced by sample concentration and the system's dynamic integration window - not unlike the FACSDiVa window extension setting. If the window is reduced, % abort would likely decrease. Also, increasing the threshold would also have the effect of better resolution between pulses and thereby decrease the abort rate. I did not explore either of these options when running and just used the default window extension and threshold. As you can see from the chart, even going at a moderate rate of 10,000 events per second yields an alarming abort rate. Going even faster results in a recovery of 50% or less. It's important to separate your ideas about % aborts on analyzers from high-speed sorters. Cell sorters have the advantage of pushing the cells through at very high velocities resulting is narrower pulses and an easier time resolving two closely related pulses. But, on analyzers, the cell velocity is much slower, resulting is broad pulses and more difficulty resolving closely related pulses. Therefore, the abort rates are typically going to be higher on slow flow analyzers, however we're not as aware of these abort rates on analyzers because we are <br />
always only concerned with frequencies of populations and not absolute yield of populations (like on cell sorters). So, 10% abort over 20,000 events per second might be reasonable, however, 10% abort rates over 10,000 eps is probably not.<br />
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For this test, I created a concentrated sample using a suspension cell line, which, at 60ul/min should yield 50,000 events per second. I then created serial dilutions from there all the way down until an expected 2,500 events per second. I ran each tube on the instrument and recorded the event rate displayed by the system's counters. </div>
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Software/Interface:</h3>
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If you've used CellQuest and FACSDiVa in your cytometry lifetime, you'll feel right at home here. There's not much to say about the software except that it works. It was super easy for me to pick up. I was shown nothing as far as how to operate the instrument, do compensation, etc... and I was able to figure it out with minimal struggle. The CytExpert software does one thing really well and that is it gives you a large, unobstructed view of your data, and just enough controls in a thin side panel to allow you to acquire data. I'm sure there are some analysis tools built in, but I don't care, I just want to acquire data, dump it into FlowJo and worry about analysis later. It gives you the ability to do automated compensation, use biexponential display, perform gating, and show stats windows. It would be interesting to see if something like Kaluza-G would ever make it onto something like this. But then again, Beckman Coulter already has 900 acquisition softwares already, what's one more!</div>
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<h3>
Performance:</h3>
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Finally the good stuff. What can I say, this thing rocks. In terms of fluorescence sensitivity, it beat the pants off of anything I've ever tested full stop. I've put a range of values together for fluorescence resolution that shows the spread of instruments I've tested. The value (called qNORM) represents the lowest number of antibodies bound that can be resolved from unstained lymphocytes. The lower the value, the better, and as you can see, the CytoFLEX, with its APD detectors and DIY lasers easily beats the average across the board. Of course I ran all the other common bead sets on this instrument. Everything I threw at it, it handled with ease. </div>
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<h4>
8-Peak Resolution at low and high flow rates:</h4>
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As you can see, resolving 8-peak beads is a cinch on the CytoFLEX pretty much across all channels. Even at the highest flow rate (240ul/min) the fluorescence resolution remains relatively unchanged, however the light scatter experiences some funky spread at the high flow rate.</div>
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<h4>
APD Voltage Optimization:</h4>
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Using a blank bead, the voltage was moved up and down the scale at appropriate intervals. The rCV was calculated on the single bead peak in each of the fluorescence channels. Using a similar test as PMT optimization, I wanted to see if the APDs behaved in a similar way. It does appear that there is a sweet spot for APD voltages that vary across parameters. This mimics PMT optimization profiles commonly seen before.</div>
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Linearity:</h4>
<div>
A pretty simple linearity test using PI stained CENs, and everything checks out as expected. However, like I mentioned earlier, linearity on this instrument has a bigger role on this instrument than others. With the CytExpert software, you can setup compensation at one set of voltages, change voltages (because it's a different cell line, or the sample is too bright, or other reason), and it will recalculate compensation based on the new voltages. This may have been (or may still be) part of the FACSVerse software, but I've never used one of those, so I'm not sure about that. Theoretically, then, you could create a set of comp tubes using non-tandem antibodies once, and then recall those comps each time, even if you've changed voltages. Anywho, linearity is great, it deviates from the theoretical line by less than 1% across the board.</div>
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhdMiwOu-kiUH5EhgqW-2ve_rJw1GDyZzPm82ek_f1JK7adOJxg0AlVde8sj5wpfwLBR5yYElYFhOS-JSjk2T_ONzTb3z2oeGsZ5kUDnL0FVfz0GGSMhuoGf8PfI0PTeGL2Kq7n4S5oQXoK/s1600/CytoFLEX_Linearity.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhdMiwOu-kiUH5EhgqW-2ve_rJw1GDyZzPm82ek_f1JK7adOJxg0AlVde8sj5wpfwLBR5yYElYFhOS-JSjk2T_ONzTb3z2oeGsZ5kUDnL0FVfz0GGSMhuoGf8PfI0PTeGL2Kq7n4S5oQXoK/s1600/CytoFLEX_Linearity.png" height="271" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">PI Stained CENs comparing the theoretical line and the actual data</td></tr>
</tbody></table>
<br />
<h4>
qNORM Resolution Comparison:</h4>
<div>
Without going to much into the methodology (because I've done it so many times before), what follows is a comparison of pretty much every instrument I've ever tested (grey boxes with quartile whiskers) with the CytoFLEX (blue circles) overlain. This metric measures the number of bound antibodies that can be resolved from unstained lymphocytes. So, lower numbers equals better low-end resolution. As you can see the CytoFLEX compares very well with all the best instruments out there. It definitely beats every instrument I own in the FITC, PE, PECy7, and APC channels. In the PacBlue channel, it's about average. I'm pretty sure this system can resolve pretty much any dim population you can throw at it.</div>
<div>
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<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhY6qELIBxtFbiyTEhIKZ_82OGa4Scxck2_4zgGqhj_BrNOoG7J2d-BS5sgQCNhlXYpFAbvba1tTN0hKwSOiTcHqQzCDoIIU007gcFOBiNicICzJbsjtdFKOqOFl8w-I-FYyYIXKh2wNq54/s1600/CytoFlex_qNORM_ComparisonChart.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhY6qELIBxtFbiyTEhIKZ_82OGa4Scxck2_4zgGqhj_BrNOoG7J2d-BS5sgQCNhlXYpFAbvba1tTN0hKwSOiTcHqQzCDoIIU007gcFOBiNicICzJbsjtdFKOqOFl8w-I-FYyYIXKh2wNq54/s1600/CytoFlex_qNORM_ComparisonChart.png" height="271" width="400" /></a></div>
<div class="separator" style="clear: both; text-align: center;">
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<h3 style="clear: both; text-align: left;">
Final Thoughts:</h3>
<div>
It's evident to me that the CytoFLEX would suit the needs of many demanding applications. There's really no questioning its performance in terms of fluorescence detection. Light scatter resolution of cell populations wasn't as good as some of my better instruments. However, small particle detection, especially using 405nm side scatter is reported to give <200nm resolution. I typically don't test small particle stuff since it's not really my thing. The fluidics seemed stable and robust for the time I had it. I ran as many cell samples as I could to see if I could clog it up or make things look bad, and other than the high abort rates at high event rates, I saw no issues from a fluidics standpoint. The software is fine for what it needs to do. I'm so entrenched in doing analysis in FlowJo that I couldn't care less if there are histogram overlays or other fancy analysis-only plots in my acquisition software. I just want it to be fast and simple to use. </div>
<div>
<br /></div>
<div>
We also don't really know about the long term reliability of the hardware components. Sure everything works fine over the course of a month, but what about a couple years or more. Will it have the staying power and uptime of a FACScan? This, I'm afraid, only time will tell.</div>
<div>
<br /></div>
<div>
But, I think the most important take home message here is that this instrument proves that flow cytometry hardware is absolutely a commodity in the eye of the consumer. As fancy as one wants to make hardware these days, no one is going to be impressed. And the fact that hardware can be made cheaply reinforces this fact. What this means is that we'll finally see a shift in focus away from over-engineered hardware to hardware that just works, but this time with a super slick user interface that people are attracted to. The future is all about software and services, and I, for one, couldn't be happier!</div>
<br />
<b>Postscript:</b> At the time of publishing, Beckman Coulter launched a new splash page with specs and a glimpse of the new exterior of the CytoFLEX. You can reach that page <a href="http://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/flow-cytometry/flow-cytometers/cytoflex/index.htm" target="_blank">here</a>. </div>
Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com2tag:blogger.com,1999:blog-8037707352330428788.post-91773394208136546752014-01-29T11:05:00.000-06:002014-01-29T11:05:48.067-06:0021st Century Learning using an Ancient Model Applied to Flow Cytometry TrainingThere are so many things I'd love to learn. I always imagined myself playing the guitar, or executing a no-hands backflip, or even writing a mobile application, but so far, I still cannot do any of those things. Of course, each of these things are certainly in the realm of possibilities for me. I'm actually somewhat musically inclined, I can do many gymnastic-type flips, and I know a thing or two about the languages of the coding world, and yet, I can't amend my CV with any of these goals. So, how might I go about learning these skills that envelope both knowledge of abstract concepts (like coding) and physical moves (like playing a guitar)?<br />
<br />
I don't know about you, but whenever I'm trying to figure something out, I default to YouTube. YouTube is great for things like this, in that you can pretty much find a video demonstrating something on any topic you're interested in. However, where things tend to fall apart is the one-way nature of YouTube. The demonstrator is broadcasting out a message that I may stumble upon years after it was uploaded and there's not a great way for me to interact with the original creator. Sure, I could leave a comment in the hopes that it'll be answered, but I'm just as likely to get an unhelpful snide remark. <br />
<br />
In fact, many models of learning these days follow a similar strategy. E-Learning is all the craze these days. Popularized by online e-learning houses such as Kahn Academy or Coursera or even on a larger scale, institutions such as University of Phoenix, e-learning promises all the bang for little to no buck, all taking place in the confines of your oversized easy-chair. But, questions remain as to how effective these programs are. I know I've signed up for courses a few times only to stop going after the second or third lecture. In some ways the information is presented as little more than a canned powerpoint presentation with some voice-over description. A step up from here is the possibility to interact, real-time with the presenter via chat or video conference. Even, with the best implementation of these technologies, remote e-learning is difficult. <br />
<br />
Let's flip this conversation completely on its head for a moment. For millennia, the way in which people learned a trade or skill or gained any sort of knowledge was through a Master/Apprentice process. The elders of the group who had the necessary experience and expertise took a young apprentice under his wing and taught him the way. If it helps, I always conjure up images of Qui-Gon Jinn teaching his Padawan Obi-Wan Kenobi in the ways of the force. You could imagine the education the apprentice received was really good, but the process was somewhat inefficient since a Master may only have a limited number of apprentices. Contrast this with e-learning and the dichotomy should be clear. E-learning may provide a highly efficient means of disseminating information, but the actual learning may be inadequate whereas the Master/Apprentice model may provide for world-class learning but is inefficient in terms of disseminating information to a large group of eager learners. <br />
<br />
To bring this conversation closer to home, how do we go about teaching the art of flow cytometry to the next generation of scientists? I would say, up until this point, the passing on of flow cytometry knowledge has favored the Master/Apprentice model. This is certainly the way I learned, and probably the way I would prefer to learn just about anything. In recent years, however, many core facilities, companies, and professional organizations have tested out the e-learning model of teaching flow cytometry. Like the e-learning trailblazers, these differ in quality from powerpoint slideshows to interactive, well-produced, and highly animated videos. We've tossed around the idea of moving towards an e-learning model at UCFlow, and what's held us back (aside from the technical complexities involved in producing something worth putting your brand on) is a core belief that we can produce better cytometrists with the more intimate master/apprentice model. <br />
<br />
The question then becomes, can we leverage modern communications technologies to make the master/apprentice model work more efficiently? Well, of course the answer is yes, otherwise I wouldn't have bothered writing this post. But, instead of describing a fictional method in overly verbose prose, I want to point you to what I think is the ultimate model of learning PERIOD.<br />
<br />
It pairs the master/apprentice model with new technologies like video conferencing, chat, hangouts, google glass, wearable tech, etc... It also gamifies the process to promote better engagement. Imagine this scenario. I know a little flow, but I'm faced with this new application. I'd really like to start doing microparticle analysis. I log into the cytometry masters portal, and search microparticles. Up pops a list of microparticle experts with various specialities and levels. For example Jane is a level 50 Endothelial MicroParticle Master, and can accept a new apprentice for the next month. She prefers to communicate via Google+ Hangouts and is in the Pacific Time Zone. I connect with Jane, learn all her tricks and tips, and then I can level-up in my knowledge of microparticle detection, bringing me to a level 10 master.<br />
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Would you like to see how this works? Luckily, this has already been launched using a different, but I'd dare to say very similar, technology - photography. The super awesome photographer, Trey Ratcliff (stuckincustoms.com) launched a brand new site, The Arcanum (thearcanum.com) that uses this exact model. Master/Apprentice, Modern Communications Technologies, Gamification. Watch the video below to see what it's all about. What I love about this is that it's visual; you're learning directly from an expert of whom you can ask all the nuanced questions you like; it uses all the latest gadgetry; and the gamification of the process makes it way more engaging.<br />
<br />
The next question I have is, Who wants to build the flow cytometry version of this with me????<br />
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<iframe allowfullscreen="" frameborder="0" height="270" src="//www.youtube.com/embed/BDL58aJSr28" width="480"></iframe>Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com0tag:blogger.com,1999:blog-8037707352330428788.post-8370426386251811042013-11-18T20:48:00.001-06:002013-11-18T20:48:05.511-06:00He said, She said, PEBCAC?<div style="text-align: right;">
</div>
In flow cytometry core facilities, scenarios such as the one that follows are commonplace.<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj90trbnTm8niURDgH383MMNjzM-teiPCOOQbGbpXsU-PKZ3dD5udPvwchC3Lzfv2hdyIAnXM1YcjWaajjINy76_ZMvLajWAYriwjBHBbFhtLN7GbI6Z8AJG37a8w80JXfyT8emnJNq1Qgy/s1600/PEBCAC.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj90trbnTm8niURDgH383MMNjzM-teiPCOOQbGbpXsU-PKZ3dD5udPvwchC3Lzfv2hdyIAnXM1YcjWaajjINy76_ZMvLajWAYriwjBHBbFhtLN7GbI6Z8AJG37a8w80JXfyT8emnJNq1Qgy/s320/PEBCAC.png" width="196" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">The first thought that comes to me<br />after a user reports a problem.</td></tr>
</tbody></table>
<blockquote class="tr_bq">
<i>An end-user is attempting to collect data, for some reason there's an issue, the end-user requests assistance from the core facility staff, some resolution is achieved, lather, rinse, repeat.</i></blockquote>
But, the interesting thing is the back and forth between facility personnel and the user. Each party is trying to figure out in what way the other party messed up the experiment. A veritable "he said/she said" ensues and eventually a resolution is achieved. The way in which the resolution comes about can take many forms depending on the level-headedness of the parties involved. However, core facility personnel are typically about as protective of their instruments and services as a mama grizzly is towards her newborn cubs. Similarly, a precocious grad student, who has spent umpteen hours preparing her samples, couldn't imagine a situation where she could have made an error. To celebrate this perennial back and forth, I present to you the 10 most common phrases (5 from each side) overheard between core facility personnel and end-users during the initial throws of an experimental/instrument mishap.<br />
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5 Most common statements<b> from core facility personnel</b> when presented with a problem by an end-user<br />
<br />
<ol>
<li>Did you try and reboot the instrument (software)?</li>
<li>Hmph, my QC beads look fine.</li>
<li>Did you filter your samples before bringing them here?</li>
<li>I don't know... everything looks pretty dead/negative to me.</li>
<li>No one else has had any problems on here today.</li>
</ol>
5 Most common statements <b>from an end user</b> when they encounter a problem at the core facility<br />
<br />
<ol>
<li>Why does this thing break every time I try and use it?</li>
<li>I had X million cells, so why did the instrument only run X/5 cells?</li>
<li>The instrument is clogged or something. The person before me didn't clean it well enough.</li>
<li>Well, will the problem be fixed soon? This data is for a grant proposal due tomorrow.</li>
<li>I hope you're not going to charge me for this.</li>
</ol>
<div>
Of course, I'm a bit biased when it comes to this scenario, so you may have your own favorite anecdotes to share. You can do so in the comments. Flame on!</div>
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<br />Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com1tag:blogger.com,1999:blog-8037707352330428788.post-43847978002157608152013-07-23T12:29:00.000-05:002013-07-23T12:29:36.633-05:0010 Tips for purchasing your next cytometerSo you've got some money to spend and you figure, heck, I do so much flow, maybe I'll just buy a(nother) cytometer. Presented here are some tips to avoid buyer's remorse, see though the marketing spin, and make an educated decision on which instrument to purchase. But, before we get into that, let me first state that I'm NOT going to make this decision for you and tell you which instrument to buy. I am merely going to provide you with the tools to make as good a decision as you can. In fact, these are the very same steps I go through whenever I'm in the market. And so, I present to you, 10 tips for purchasing your next cytometer.<br />
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<b>#1. Define your needs.</b> What are the applications you will run on this system? How many parameters (realistically) do you run on average? How many parameters will you run in the near-future? Are there any specialty dyes you run? Do you prepare samples in tubes or plates? At what event rate do you run your samples?<br />
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<i>Example: I have some projects in mind which require 8 - 10 fluorescence parameters. At 3 parameters per laser, I probably need a 3-laser system minimally. I like to stain/run my cells in a 96 well plate, so a plate sampler option is needed. My experiments typically involve immunophenotyping rare subsets, so I collect 10^6 cells at rates of about 15,000/second. I don't need to sort.</i><br />
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<b>#2. Query the end-users.</b> If others in the lab or core facility will use the instrument, ask them the same questions as in #1.<br />
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<i>Example: Another user in the lab does a lot of screening of her mCherry transfected cell lines. She doesn't collect a lot of cells, but screens many samples. She would require a yellow/green laser for excitation, and fast 96-well sampling capabilities.</i><br />
<br />
<b>#3. Refine your needs.</b> Combining the information you learn from steps 1 and 2, you should be able to refine the needs for this instrument. Annotating this list, and possibly triaging needs and wants will be very helpful at this point. Of course you are working within a budget, so you'll certainly want to keep that in mind as you survey the market.<br />
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<b>#4. Survey the market.</b> You probably already have an idea of the "big" players in the market, but even if you didn't, simply typing the query "flow cytometer" in your browser brings up 9 different instrument manufacturers within the first two pages of a Google search. You can follow these links, collecting information on the various instruments. For something like this, I like to use an electronic note taking application like <a href="http://evernote.com/" target="_blank">Evernote</a> to keep everything together, and make notes as I go through the process. For those web sites where information and materials are not easily accessible, sending an email to a local sales representative will get you all the marketing materials you could ever ask for.<br />
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<b>#5. Learn how to read marketing materials.</b> Speaking of marketing materials, there are a few things you should be aware of. Beyond the very basics (lasers available, number of detectors, etc...), much of what you see in your average cytometer specification sheet is useless information. It's basically a list of values for meaningless metrics that MUST be put into the materials to match what the competition is stating. For example, the ever-present detection threshold of FITC and PE. Most all spec sheets will include something like an MESF Detection Threshold of <150 for FITC and <100 for PE. This means absolutely nothing in terms of how well the system will work for your applications let alone how other colors will fair. You'll also see outrageous specifications for event rates, like 100,000 events per second! Lastly, and probably my favorite, is the panel of histograms showing the resolution of multi-intensity hard-dyed beads (e.g. 8-peak Spherotech beads). You can pretty much ignore all this information, and focus on the things that matter. How many lasers? How many detectors? Can you upgrade the system in the field with more lasers/detectors? Is there a multi-well sampler? etc...<br />
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<b>#6. Create the matrix.</b> By now, you have a list of needs/wants, and you have a bunch of marketing materials. Put it all together in tabular format.<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEivQsCz_5bjnho4eCqSRCrx6xD_El0N5PLS9jfIY2xQdkVkl1QCcsVsf5ECaBRHl8pzR-dt2qeA1zFB7PfIyZGpbQ2tUZ2Slcb8a0Tk-Q1Pp71jSmZ9YdESgR_J4Ov-btsNVE3m3UPJBZiz/s1600/InstrumentComparison.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="127" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEivQsCz_5bjnho4eCqSRCrx6xD_El0N5PLS9jfIY2xQdkVkl1QCcsVsf5ECaBRHl8pzR-dt2qeA1zFB7PfIyZGpbQ2tUZ2Slcb8a0Tk-Q1Pp71jSmZ9YdESgR_J4Ov-btsNVE3m3UPJBZiz/s400/InstrumentComparison.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Fictitious Instrument Comparison Chart, with the 3 contenders.</td></tr>
</tbody></table>
<br />
<b>#7. Gain hands-on experience for the top 3 contenders</b> - make sure the OEM knows the fate of the sale hinges on the success of the hands-on demo. Run your battery of tests that matter to you, evaluating the results, as well as ease-of-use, software, UI, hardware. Simply staining your favorite panel of antibodies and running it on the instrument will give you a TON of information as to how these cytometers stack up. Running real samples (not just beads) is an absolute must.<br />
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<b>#8. Take to the social network</b> (and take everyone's opinion with a grain of salt). Useful things that can come back from the community include; recurring hardware/software failures, maintenance issues, service issues, responsiveness, etc... For any negative responses that come up, make sure to bring these to the attention of the manufacturer (respecting people's confidentiality, of course) and ask for a response. Get everything in writing. No phone conversations!<br />
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<b>#9. Negotiate the purchasing terms with multiple companies.</b> Make sure the sales representative is aware of their competition. Aside from asking for the best possible price, discuss other value added options. For example, an extension of the warranty, free training slots, discounted multi-year service agreement, free shipping, free upgrades (higher powered lasers, multiwell samplers, extra emission filters, next version of software). Get everything in writing, no phone conversations (did I mention that already)!<br />
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<b>#10. Take advantage of year-end discounts.</b> If possible, time your negotiations and purchase with the end of the company's fiscal year. You'd be surprised what sort of deal you can get if the company is close to reaching their target for the year. <br />
<br />
<b>BONUS Tip:</b> Don't be afraid of venturing away from the "big companies." When dealing with newer companies and newer technologies, getting cutting-edge hardware can be a double-edged sword. Although you may be able to get a deal on price, make sure there are some protections in place that allow you to get future upgrades or revisions to problematic hardware for free. At the very least, you should be able to get a percentage of your money back if it's a total failure. Again, get it in writing up front.<br />
<br />
So there you have it. I think if you keep these common sense tips in mind when purchasing your next cytometer you won't be disappointed. Got any other tips that have helped you make the right purchasing decision? Why not leave a comment below.<br />
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<br />Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com0tag:blogger.com,1999:blog-8037707352330428788.post-50180557744979644852013-06-06T19:48:00.000-05:002013-06-06T19:48:12.851-05:00A Cell Sorter in Every Lab. Can Core Facilities Survive?<div class="separator" style="clear: both; text-align: center;">
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgGesp_ViigGM8ls4SS1O3kpAi8kyR-Lqy2IAK_qgLKsdB8rvUPz8qw1cBhnaE_vlzIH7PS6MBkhkR6_v8aHaRBDGm0IwJVe2OcAoasBuiJX3r4IwEJOlGaNXqbcribN2XinpeJ9ozNgtJD/s1600/keep-calm-and-go-with-the-flow-132+(1).png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgGesp_ViigGM8ls4SS1O3kpAi8kyR-Lqy2IAK_qgLKsdB8rvUPz8qw1cBhnaE_vlzIH7PS6MBkhkR6_v8aHaRBDGm0IwJVe2OcAoasBuiJX3r4IwEJOlGaNXqbcribN2XinpeJ9ozNgtJD/s400/keep-calm-and-go-with-the-flow-132+(1).png" width="342" /></a></div>
It happens in every industry across all times - What initially requires sophistication and expertise becomes simpler and more accessible to the masses and the former "experts" feel threatened and rail against the advancements. For many years, Flow Cytometry core facilities cornered the market on ALL cytometry taking place at an institution. As instruments became easier to use, some facilities allowed their users to begin operating the analyzers unassisted. Affordable flow cytometers first came to market in the form of the <a href="http://www.millipore.com/flowcytometry/flx4/flow_cytometry_guava" target="_blank">Guava</a> with some success, and then, in late 2006, Accuri exploded on the scene with it's affordable, easy-to-use <a href="http://www.bdbiosciences.com/instruments/accuri/index.jsp?WT.ac=FP_Accuri" target="_blank">C6 analyzer</a>. Undoubtedly, these instruments were marketed to individual investigators seemingly bypassing core facilities altogether. <br />
<br />
The last stronghold of core facilities seemed to be cell sorting. Alas, these instruments are sufficiently complicated as to assure even the most skittish of core facility technologist. That was, until easy-to-use sorters became more available. The <a href="http://www.bdbiosciences.com/instruments/facsaria/index.jsp" target="_blank">FACSAria (BD)</a> was marketed as the first bench-top cell sorter capable of doing everything its more complicated predecessors could do. That didn't pan out so well, much to the chagrin of BD. However, after multiple iterations of the FACSAria, as well as other, easy-to-use cell sorters, we're on the cusp of a turning point in cell sorting, much like that day in 2006 when Accuri launched its C6. In fact, here at the University of Chicago, we've jumped on this bandwagon with both feet, adding the <a href="http://www.bio-rad.com/prd/en/US/LSR/PDP/MC3PU4E8Z/S3-Cell-Sorter" target="_blank">BioRad S3 Cell Sorter</a> to our group of cell sorters. In addition to training users to operate the FACSAria's, we now can spend much less time training users to sort on the S3. For a user who is familiar with the general operation of a flow cytometer, we can get them proficient on the S3 in less than a half hour. Now, roughly one third of simple sort clogging up the FACSAria schedules can be done on the S3 with no increase in facility personnel and minimal increase in facility operating expenses. Win/Win/Win!<br />
<br />
<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgsLR7RMTA_M2IrXqmAXThE0iZIUY_q9gK6MwPta_wrsLy4idR7H346cT_0mAeWBSd16GBLR0fUbbiRnQNXWGF4c-_8jZyrJStrM5BCQ1E_SbDfgZkN1im1_bzDhLY2ytGDSUQonw3IA8do/s1600/Took_er_Jobs.jpg" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="180" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgsLR7RMTA_M2IrXqmAXThE0iZIUY_q9gK6MwPta_wrsLy4idR7H346cT_0mAeWBSd16GBLR0fUbbiRnQNXWGF4c-_8jZyrJStrM5BCQ1E_SbDfgZkN1im1_bzDhLY2ytGDSUQonw3IA8do/s320/Took_er_Jobs.jpg" width="320" /></a>However, some of us in the field may feel like this is yet another assault on our job security. "If I don't control the sorting, what will I do all day." I think this view is extremely myopic. From an economic standpoint, it's always better to get something for nothing than to have to put real resources into doing it. When you think about it more closely, if we don't have to expend resources into operating cell sorters, we can focus those resources on other important things. I mean, it's sort of silly to have a trained professional sitting in front of a cell sorter drawing regions around GFP+ cells. These types of sorts can easily be transferred back to the user once she is trained to operate a basic cell sorter. This frees up time for the experts to focus on the cutting-edge stuff or development work. <br />
<br />
This whole discussion then turns to a more philosophical discourse on what the role of resource core facilities will be in the future. It's time to pivot, folks! Gone are the days of a facility with 1.25 FTE's per cell sorter, staring at dots popping up on the screen all afternoon. The business side of me wants to focus less on hardware recharge and more on services. Why be a warehouse of hardware, when you can be the (more lucrative) service center. Let's assume, for a moment, that cell sorters like the S3 or <a href="http://www.sony.net/Products/fcm/products/sh800/index.html" target="_blank">Sony's SH800</a> become so solid and affordable that many labs decide to buy their own. Facilities focused solely on hardware recharge revenue will quickly spiral towards obsolescence. Facilities focused on services will already have other revenue streams to compensate for the lost hardware recharge. Allow me to illustrate with a few examples.<br />
<br />
<b><u>Sample processing as a service (SPaaS):</u></b> An investigator has an idea for an experiment on a cohort of patients seen in the clinic. We, as a core facility can coordinate pick-up of blood tubes from the phlebotomist, process the blood, bank the plasma and cells, and then stain the cells using standard panels and perform cytokine bead assays, microparticle analysis, or other assay on the plasma. Translational applications like this are not only becoming more and more common, but many times those investigators in the best position to do these studies do not have large research labs set up to do this themselves. Since we're not sitting in front of the FACSAria 8 hours a day, we now have time to develop these panels, and market this service.<br />
<br />
<u style="font-weight: bold;">Internal Instrument Service and Maintenance (IISM):</u> Investigators start purchasing their own analyzers/sorters, however, they may not have the skill, time, or resources to perform routine service and maintenance. We, in the core facility already have the know-how to fix many of the issues on these instruments, and we have the SOPs in place to perform the necessary maintenance and quality assurance. Instead of paying a service contract fee to BD AND having to do all the routine maintenance themselves, the investigators could contract the flow core facility to perform maintenance and service. The core facility can, in turn, take out a self-insurance policy to maintain the instrument and recover actual costs. The more instruments the facility can perform this service on, the better insulated you can be against catastrophic incidents. <a href="http://ucflow.blogspot.com/2011/11/cytometer-service-contract-or-self.html" target="_blank">This is essentially the model I've been using</a> in my own lab; Budget 50% of the cost of a service contract, and pay for service calls as needed. In the 15+ years I've been around, we've never lost money. This works only because the number of instruments is sufficiently large. The use, operation, and initial investment of the hardware took place completely outside the core facility. The investigators' staff and students need training? We can do that! They want to assure the instrument is performing optimally? We can do that! They need someone to come fix the instrument because it's not working? We can do that! Or, if we can't, we call the OEM and pay for a service call.<br />
<br />
Of course, there will still be other technologies associated with a traditional core facility that will still follow the normal hardware use recharge model. High-end cytometers/sorters, imaging cytometers (a la the ImageStream), mass cytometry (CyTOF), etc... However, we can anticipate these technologies following suit. Soon, the CyTOF will be super easy to run, and imaging technologies will be simpler and simpler (e.g. the FlowSight). As these technologies become commoditized like analyzers and now cell sorters, it's going to be service-focused facilities that will stand the test of time.<br />
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<br />Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com1tag:blogger.com,1999:blog-8037707352330428788.post-32033596513979194692013-03-11T10:35:00.000-05:002013-03-11T10:47:25.564-05:00ABRF 2013 Recap - A flow cytometrist's take.<span style="background-color: white; line-height: 16.796875px;"><span style="font-family: inherit;">On the ABRF <a href="http://www.abrf.org/index.cfm/page/home/mission.htm" target="_blank">about page</a>, it states, "The Association of Biomolecular Resource Facilities is an international society dedicated to advancing core and research biotechnology laboratories through research, communication, and education." But, if you know anything about ABRF, you're likely involved in a molecular biology-based core facility, namely a genomics, proteomics, or related facility. I guess I always knew about ABRF, their meetings, and what they were about, but somehow it never really interested me much. I mean, anything with the word "biomolecular" in it gives me not-so-pleasant flashbacks to biochemistry classes. So you can see why a guy who spends all his time working with whole cells might not take a second look at marketing materials from ABRF. However, it seems as if the tide has shifted.</span></span><br />
<span style="background-color: white; line-height: 16.796875px;"><span style="font-family: inherit;"><br /></span></span>
<span style="background-color: white; line-height: 16.796875px;"><span style="font-family: inherit;">What piqued my interest this year was some interesting movement in two of the ABRF research groups that had formed in recent years. You see, unlike other societies which may only focus on annual meetings, <a href="http://www.abrf.org/index.cfm/page/research_groups.htm" target="_blank">ABRF has interest groups</a> that form with the intent of doing research projects. A core group of ABRF members with common interests (e.g. flow cytometry) may come together and propose research projects to work on. ABRF supports these efforts by providing the necessary sponsorship. A newly formed <a href="http://www.abrf.org/index.cfm/group.show/FlowCytometryResearchGroup(FCRG).76.htm" target="_blank">Flow Cytometry Research Group</a> (FCRG), and a recently revived<a href="http://www.abrf.org/index.cfm/group.show/AntibodyTechnology(ATRG).67.htm" target="_blank"> Antibody Technology Research Group</a> (ARG) were working on some projects that seemed really interesting and very pertinent to what I do. Seeing as I now <a href="http://ucflow.blogspot.com/2012/01/introducing-cytometry-and-antibody.html" target="_blank">share my time between our antibody production and flow cytometry cores</a> you can probably guess why I'm excited by these two research groups. Combining this with the general core facility management stuff that's always happened at ABRF pretty much made up my mind about attending this year...and I'm glad I did!</span></span><br />
<span style="background-color: white; line-height: 16.796875px;"><span style="font-family: inherit;"><br /></span></span>
<span style="background-color: white; line-height: 16.796875px;"><span style="font-family: inherit;">Sure there were some interesting talks about exome sequencing and insanely parallel westerns, and even the need to foster convergent technologies in order to make inroads into cancer research, but the real highlights came at the FCRG and ARG meetings. The ARG group had been working on a modified immunization strategy to both increase the initial immune response as well as prolong that response in order to trick the immune system into making antigen specific, antibody secreting B cells. One might not think of cutting edge technology when talking about novel monoclonal antibody production, but they had some interesting ideas. For example, using CpG's in combination with a standard adjuvant (e.g. Freunds) when an antigen doesn't seem to be eliciting a good response. This would be hugely important information for our Antibody facility. The FCRG also had some interesting data surrounding the ill effects on cell function after cell sorting. Everyone has their anecdotes about sorting at high pressure vs. low pressure, or on a jet-in-air sorter vs. cuvette sorter, but there's not much data out there in a well-controlled experiment. Again, hugely important information. </span></span><br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhAo1o9_Da7u0zs4xatQb1u3E8Mpr7yB9egbrG8sjRnHCS3jRFIlOhqTGHMzZ7PPzzvjypY-GiCuPFtbHumQTkwTwX7uL2jmBS3AU-7zENkKY21yOVqQLNoJ5f4DK-sl1bboB3WOmnXCeZx/s1600/ABRF+Award+Herzenbergs.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="508" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhAo1o9_Da7u0zs4xatQb1u3E8Mpr7yB9egbrG8sjRnHCS3jRFIlOhqTGHMzZ7PPzzvjypY-GiCuPFtbHumQTkwTwX7uL2jmBS3AU-7zENkKY21yOVqQLNoJ5f4DK-sl1bboB3WOmnXCeZx/s640/ABRF+Award+Herzenbergs.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">2013 ABRF President, David Friedman presenting Lee & Len Herzenberg with the ABRF Award</td></tr>
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<span style="background-color: white; line-height: 16.796875px;"><span style="font-family: inherit;"><br /></span></span>
<span style="background-color: white;"><span style="font-family: inherit;"><span style="line-height: 16.796875px;">Another interesting draw for me was the fact that <a href="http://en.wikipedia.org/wiki/Leonard_Herzenberg" target="_blank">Drs. Lee and Len Herzenberg</a> were being honored with the ABRF Award, and anyone hanging around with any involvement with flow cytometry got to get in a group picture with the Herzenbergs. I have to admit being a little star-struck around them. I really wanted to ask them for an autograph, but I didn't. We were treated to some behind-the-</span></span><span style="line-height: 16.796875px;">scenes</span><span style="font-family: inherit;"><span style="line-height: 16.796875px;"> photos of the early days of flow cytometry. And, it was also interesting to note how this development coincided with the </span></span><span style="line-height: 16.796875px;">development</span><span style="font-family: inherit;"><span style="line-height: 16.796875px;"> of the first personal computers. In fact, Lee spoke about some of the first computer programs built by Wayne Moore and Dave Parks. Probably the best part were the pictures of the 1970s versions of Wayne Moore and Dave Parks... Yeah, they look pretty much the same.</span></span></span><br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh7IIawrsmkbxePW6z3UMd6PcyO_AoB8P0BghV0usVaYUFEIk9Vlim_uWHRNiAN5yzfUWxhcPDkfDs1WBrgDfmnTnn1MvsaV6jEfGdxu8txJzEJrh_yHgWRGPHPIJKIOw9S7K0zjbe4wmYa/s1600/ABRF+FCRG+and+Herzenbergs.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="442" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh7IIawrsmkbxePW6z3UMd6PcyO_AoB8P0BghV0usVaYUFEIk9Vlim_uWHRNiAN5yzfUWxhcPDkfDs1WBrgDfmnTnn1MvsaV6jEfGdxu8txJzEJrh_yHgWRGPHPIJKIOw9S7K0zjbe4wmYa/s640/ABRF+FCRG+and+Herzenbergs.jpg" width="640" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Member of the Flow Cytometry Research Group posing with the Herzenberg's following their tandem ABRF lecture</td></tr>
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<span style="background-color: white;"><span style="font-family: inherit;"><span style="line-height: 16.796875px;"><br /></span></span></span>
<span style="background-color: white;"><span style="font-family: inherit;"><span style="line-height: 16.796875px;">I have to say, I was quite impressed with the meeting in general. The company that assists ABRF in putting on this show does a stellar job, and since I'm sort of involved in putting on shows on a much smaller scale, I definitely pick up on those things. It definitely had the grandeur of a CYTO meeting, but you didn't feel completely lost in the crowd. The biggest let down for me was the exhibitor area. Not that it was poorly set up or anything like that, it's just none of my people were there. One </span></span><span style="line-height: 16.796875px;">measly flow cytometry exhibitor was there, and only because they're brand new in flow cytometry and also do some stuff in the molecular biology area. Thanks Bio-Rad.</span><span style="font-family: inherit;"><span style="line-height: 16.796875px;"> Other than that very minor demerit, it was a great conference, even for a flow guy. I'll predict that the next time CYTO is in Europe, the US cytometry contingent will flock to the ABRF meeting. You heard it here first, folks! Actually, I heard it first from someone else at the meeting and am just shamelessly taking credit for the idea.</span></span></span><br />
<span style="background-color: white; line-height: 16.796875px;"><span style="font-family: inherit;"><br /></span></span>Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com1tag:blogger.com,1999:blog-8037707352330428788.post-24450653905463357252012-12-13T13:33:00.001-06:002012-12-13T13:33:43.100-06:00Long live the Listserv; Death to the Listserv.Good ole listservs (or listserves or list serves, if you will). A staple in the academic's toolbox of communication technologies. But the question is, how useful are they in today's hyper-connected, socially-networked, always-on environment? Here we'll touch on a few pros/cons as well as get you plugged in to cytometry networking hubs online. <br />
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<b>Why I love Listservs:</b><br />
<br />
<ul>
<li>Let's face it, we all live in our inboxes, right? So, what better place to funnel all your networking than via e-mail? It's fast, easy-to-use, and is easily accessible, especially with today's smart phone install base. </li>
<li>It's a pretty good networking tool. Regarding the listserv's involvement in Cytometry, there has been no better platform to allow end-users to interact with field experts. Of course, we cannot mention the words 'cytometry' and 'listserv' in the same breath without pointing you to the preeminent spot for networking, the <a href="http://www.cyto.purdue.edu/hmarchiv/index.htm" target="_blank">Purdue Cytometry List</a>. For years (over 20, now) the Purdue List (as it's commonly referred to) has allowed cytometry professionals to interact and network.</li>
<li>Being an active Listserv participant also gains you exposure, which can lead to new opportunities. </li>
</ul>
<br />
<br />
<b>Why I wish Listservs would crawl up into a little ball and be subjected to a slow painful demise:</b><br />
<br />
<ul>
<li>Poor search leads to lazy researchers. The fact that it can be difficult to search listservs inevitably results in people asking the same questions that were answered already on the list. Long-time list participants may respond with not-so-pleasant remarks to such a query. This sometimes even leads to a discussion on the merits of re-visiting previously answered questions, when we should be discussing the original question itself. </li>
<li>"Out-of-office" replies...need I say more?</li>
<li>The "Good Samaritan Effect." People who may know the answer to a posted question may pass by without helping because they assume someone else will help them out. </li>
<li>Too broad or too diverse. Listservs typically cannot be broken down into categories, allowing people to focus in on a specific area of the listserv's main topic.</li>
<li>No inline rich media. Depending on the listserv, you may not be able to attach documents, pictures, movies, etc... Try and explain your gating strategy only using words...It's not fun.</li>
<li>Wikipedia tells me that <a href="http://en.wikipedia.org/wiki/LISTSERV" target="_blank">LISTSERV</a> was developed in 1986. Nineteen Hundred and Eight Six!!!!! Do you know how long ago that was? It was the last time the <a href="http://en.wikipedia.org/wiki/1986_Chicago_Bears_season" target="_blank">Bears won the Superbowl</a> (OUCH!).</li>
<li>Do we really need to be shuffling around emails to 4000 people on a listserv? Answer: No.</li>
</ul>
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<b>21st century tools for a 21st century technology</b></div>
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<b><br /></b></div>
<div>
Obviously, I wouldn't bring you here unless I had some alternatives to offer. And, you can probably already guess a few of the punchlines, but allow me to state the obvious. <u>SOCIAL</u> <u>NETWORKING</u>. LinkedIn, Facebook, Google+, and Twitter have demonstrated the power of social networking platforms. Already, there is a substantive presence of cytometry on each of these platforms; some of them are actually quite fruitful. These tools allow for:</div>
<div>
<ul>
<li>Sharing rich media inline with text to create better communication of ideas.</li>
<li>A better sense of interactivity of the group instead of a one-to-one interaction.</li>
<li>Fantastically good search tools for finding exactly the information you need</li>
<li>Using email notification settings, you have the ability to interact as much or as little as you'd like.</li>
<li>Speaking of email. Many of these services allow you to fully interact with the group using the email interface, if that's more your style.</li>
<li>Strong sense of community - Via avatars and in-depth profiles, you're able to build better relationships with colleagues.</li>
<li>Expand your interaction with people on the fringe since they're already using these networks for other (personal and professional) purposes.</li>
</ul>
<div>
<b>So, what's out there?</b> </div>
</div>
<div>
<br /></div>
<div>
Well, if you just do some searches, you're bound to find groups online. To point out one near and dear to me, I'll plug the fledgling <a href="http://goo.gl/fVWQe" target="_blank">Google+ Cytometry Community</a> (for which I'm one of the moderators). Google+ as a platform, is becoming quite full-featured in this regard, and the potential to have a very interactive online community is strong. Of course, the fact that it's backed by powerful search and a host of integrated tools (Drive, Gmail, Picasa, etc...) makes it, de facto, a force to be reckoned with. It is becoming more and more clear that tools like Google+ are most definitely the future, and antiquated platforms like listservs are (slowly) in decline. My advice to you? Get out there and start interacting. Why not hop over to Google+ and grab yourself an account (if you don't have one yet) and then stop by the Cytometry Community to say hi!</div>
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Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com0tag:blogger.com,1999:blog-8037707352330428788.post-22802706258171127712012-10-17T15:01:00.002-05:002012-10-17T15:03:17.780-05:00Life Technologies Attune Cytometer... Deja VuIt wasn't so long ago that I <a href="http://ucflow.blogspot.com/2011/03/getting-in-tune-with-attune.html" target="_blank">blogged my impressions</a> of the newly released Attune Cytometer from Life Technologies. Although overall, the system seemed pretty good, I did find some disheartening issues with both the hardware and software. There's no reason why this instrument shouldn't be one of the best pieces of hardware on the market, regardless of price-point. The entire premise of acoustic focusing should lead to excellent fluorescence resolution. So, when Life Technologies approached me again to give the Attune another look, I obliged. The reason for the poor performance the first go around? Poor alignment and a very generous install specification. With some of those issues taken care of, I was able to spend a few months with both the Blue/Red laser configured system and the Blue/Violet system. I'll reference back to the original article in various places, and highlight some of the improvements I've seen with the systems.<br />
<br />
<b>Fluidics:</b><br />
<br />
Much of the fluidics system has remained unchanged for the most part. A few of the biggest issues from the prior testing have been addressed specifically point #8 where there seemed to be some fluctuation of the fluorescence signal after a re-draw of the sample syringe. That no longer happens and is not an issue. The other issue is the return of unused sampled once you've acquired enough events. Now, at the end of a run, you can enable a script to return the unused portion of the sample to your tube. I'll also mention the startup and shutdown procedures here as well. They've really done it right on this system. You can start and shutdown the fluidics without having to log in (useful for usage tracking in a core facility). Also, the system turns itself off after the shutdown is complete, which is nice at the end of the day. Lastly, their performance tracking QC module works well and is very informative, yielding Levy-Jennings-type plots for all your parameters as well as giving you an overall pass/fail (similar to CS&T in DiVa).<br />
<br />
<b>Software:</b><br />
<b><br /></b>
If you've used DiVa, you've used the Attune software. The software was pretty snappy overall, and handled large data files (>100,000 events) without hesitation. There's still a lag switching between tubes while you're waiting for the fluidics to finish its process. It's long enough for you to notice it, but not so long that it becomes annoying. It's times like this however, that I wish I would have had the 96 well loader on the system. Also, the plot scale needs to be changed. Right now, they're displaying a 7-log scale of which the first 2 logs are pretty much useless. In setting up an unstained control, you end up putting your cells between the 2nd and 3rd decade anyway, so there's no reason to have empty white space at the bottom of your plots. This only adds to a user's confusion on how to optimally set up their voltages.<br />
<br />
There is no noticeable differences in <b>Optics</b> or <b>Electronic</b> other than the introduction of a Blue/Red laser configured instrument, which is built as a 4-2 configuration.<br />
<br />
<b>Data:</b><br />
<br />
As far as testing goes, I focused on resolution tests I had run previously on the system, which utilizes stained capture beads. I was working on a further development of this technique which has resulted in a quantitative metric called qNORM. The qNORM can be defined as the minimum number of antibodies bound to a cell that can be resolved from unstained lymphocytes. For example a qNORM value of 1000 would mean that this channel on this instrument could resolve a cell type that was stained with as few as 1000 antibodies from the unstained cells. In testing the Attune systems, I used a few other instruments in and around the flow facility for comparison's sake. The first table below summarizes the instruments used (including the laser powers and the vintage/health of the specific instrument tested). The second bar graph shows the qNORM values for the different instruments for 5 fluorochromes. Please note: lower qNORM values = better resolution.<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEghlOD8-E_SWxcv1KqgjbS98JDWI69APo6PnU9BmTo7MK1kfaW6lXfzO0AbVZvhzvP5c3Ixzy5sszTus73zKGO5UBMSgpqSPr2fsXyOQxjxfDA3xS8q7vXSZdccthQtLqXnt9uH2TtW0iKb/s1600/InstrDescriptionqNORM.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="126" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEghlOD8-E_SWxcv1KqgjbS98JDWI69APo6PnU9BmTo7MK1kfaW6lXfzO0AbVZvhzvP5c3Ixzy5sszTus73zKGO5UBMSgpqSPr2fsXyOQxjxfDA3xS8q7vXSZdccthQtLqXnt9uH2TtW0iKb/s400/InstrDescriptionqNORM.png" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Instruments at UCFlow tested for the qNORM characterization</td></tr>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuUbOmmjuCAlNMFlVU4bf6HAcYG3G2zYdz8c7tGfiT-bv9LQhKy_tY6G6e_9NzzmLGMD3MYYJSOAA_QFEMbk3sA69rpyEzCWDtBqslz2c5V1iIdAmZ1slsaTmFonhrOsLDxOlQdZml0NMh/s1600/qNORMData.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="300" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuUbOmmjuCAlNMFlVU4bf6HAcYG3G2zYdz8c7tGfiT-bv9LQhKy_tY6G6e_9NzzmLGMD3MYYJSOAA_QFEMbk3sA69rpyEzCWDtBqslz2c5V1iIdAmZ1slsaTmFonhrOsLDxOlQdZml0NMh/s400/qNORMData.jpg" width="400" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">qNORM values for 5 channels on the respective instruments. Error bars are calculated from 4 separate experiments.</td></tr>
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What we can take home as far as the Attune's performance on these tests is that it falls in line with its cohort on some of the channels (similar in FITC to a FACSCanto and MACSQuant), but does surprisingly well in other channels (as good as the Gallios or Fortessa in the PECy7 and APC Channels). You'll notice an anomaly in the PECy7 performance of the BV Attune compared with the BR Attune. This is due to a suboptimal filter on the BV for PECy7 (a 650 LP instead of a specific PECy7 filter such as a 780/60 BP) and a non-red sensitive PMT in that channel. Both of these deficits can be easily fixed by swapping in a appropriate filter when using that color or using a more red-sensitive PMT. <br />
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<b>Final Thoughts:</b> The units I evaluated this time around performed much better on all the tests run. The most remarkable feature of the Attune is its ability to perform on par with the more expensive cytometers in some of the channels AND retain that level of resolution at much higher flow rates. I said it before, and I'll say it again, "There's no reason why this instrument shouldn't be one of the best pieces of hardware on the market regardless of price-point", and if they continue to develop the platform, specifically working on the software issues highlighted above, I think it could easily take that crown.Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com1tag:blogger.com,1999:blog-8037707352330428788.post-17418242621839542672012-08-28T21:50:00.001-05:002012-10-31T14:42:39.639-05:00Propel Labs Avalon: A first look.N.B. The Avalon cell sorter is now the S3 cell sorter distributed by Bio-Rad. It's the same hardware/software, with a new color scheme to match the Bio-Rad colors. You can find the product's new page here: http://www.bio-rad.com/prd/en/US/LSR/PDP/MC3PU4E8Z/S3-Cell-Sorter<br />
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I recently had the opportunity to check out the highly anticipated <a href="http://propel-labs.com/products/avalon/" target="_blank">Avalon cell sorter</a> from little-known <a href="http://propel-labs.com/" target="_blank">Propel Labs</a> (Ft. Collins, CO). Little known, that is, until this year's CYTO conference where they unveiled the new 2-laser, 4-color bench-top cell sorter. This made such a huge splash at CYTO even competitor vendors couldn't help but check it out. After the dust settled from the whirlwind tour, would the Avalon prove to be a true, easy-to-use, bench-top cell sorter? Read on for the answer.<br />
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Do you remember when the original FACSAria was announced? It was billed as so easy to use, you could train all your users to run the sorter themselves. BD was forced to quickly retract those marketing talking points as things like "<a href="http://ucflow.blogspot.com/2009/02/aria-to-aria-ii-upgradepainful-but.html" target="_blank">Nozzle-gate</a>" came to light. Since then, there have been a few attempts at "user-friendly" sorters, but all them required too much hardware manipulation by the user. So, you could imagine I was pretty skeptical when the new Avalon was touted to be easy-to-use. As such, my evaluation of the Avalon focused primarily around this notion that I could train any of my users, who possessed a certain level of flow cytometry know-how, to sort their cells on this instrument with minimal instruction. I was NOT disappointed. Secondarily, I wanted to test the basics, like sorting speed, yield, purity, and to a lesser degree fluorescence resolution. Let's begin.<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEinbnaYWup7pgUl5cMMKzEx_j7Wxukhh3Ijt1iAVEHjW7DyHPKkRJi9WWPgSdVgRkZaS4aXMmjI2XLsipZEkWmozcrKhjB9RMjjMcAOZaxS-76GM9Smf7sNb5-idjt3SencjScCps86yFTi/s1600/Avalon.png" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="194" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEinbnaYWup7pgUl5cMMKzEx_j7Wxukhh3Ijt1iAVEHjW7DyHPKkRJi9WWPgSdVgRkZaS4aXMmjI2XLsipZEkWmozcrKhjB9RMjjMcAOZaxS-76GM9Smf7sNb5-idjt3SencjScCps86yFTi/s320/Avalon.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">The Propel Labs Avalon Cell Sorter</td></tr>
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First, I will say the instrument is pretty nice looking. At 70 cm wide x 65 cm tall x 65 cm deep, it's bigger than a breadbox, but not much bigger. And the best part is, what you see here in the image to the right is the ENTIRE instrument, no wet cart, no external power supplies, no nothing. Everything is contained within that footprint. Behind the larger grey door on the front houses the sheath (more on that in a sec), waste tank, and DI water tank (used for shutdowns). And here's the kicker; You can load a tank of 8X sheath fluid, and it dilutes your concentrate to 1X on-board using the DI water. Now, there's even less of a chance of introducing contamination when making up your 1X PBS for your cell sorters. <br />
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To test the system's sorting ability, I ran a sample using my normal cell sorting specification. The spec we use at UCFlow is: Event Rate = Frequency/4, and sorting a 5% population, the efficiency should be >80% and the purity should be >98%. This spec is mostly governed by physics and poisson statistics, so if an instrument cannot pass this spec, then there is something else going on (too high of a hard abort rate, too slow of an electronics processing system, etc...). Below are the results (analyzed in FlowJo, of course). As you can see, the Avalon easily handled this test. N.B. The sample was fixed/no-wash peripheral blood stained with CD3, CD4, and CD56, which is why the background is pretty high and the separation is not spectacular. This is also why the post-sort populations actually look better; we've essentially 'washed' the sample by diluting it in the collection media. You'll also note here the somewhat standard droplet formation values: 30 PSI, 100um tip, 38KHz. There are 3 modes for sorting, Purify, Enrich, and Single Cell, and in Purify or Enrich, the sort envelope is 1/2 droplets.<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg9NyZ8wzQuayJTWgUomPdItTHCvKhXrn6jsFblXyoxVgF0iV4ArJtIibTvPWPcGEkBGMhyphenhyphen_eag0B6DFKUdIo_wBZh2EI_qK9zNMIvzuCNd6PH0-cwDYF2q61tiCJTnMsUCi0ihqvmG6G_o/s1600/Eff_Pur_atMaxSpeed.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="400" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg9NyZ8wzQuayJTWgUomPdItTHCvKhXrn6jsFblXyoxVgF0iV4ArJtIibTvPWPcGEkBGMhyphenhyphen_eag0B6DFKUdIo_wBZh2EI_qK9zNMIvzuCNd6PH0-cwDYF2q61tiCJTnMsUCi0ihqvmG6G_o/s400/Eff_Pur_atMaxSpeed.png" width="365" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Sorting test using 3-color staining of fixed/nowash Peripheral Blood.</td></tr>
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The 100mW 488nm and 561nm lasers are plenty powerful to resolve most populations. This, combined with a careful selection of Semrock filters, and an in-house developed collection lens makes the Avalon no slouch when it comes to fluorescence resolution. The requisite 8-peak bead data is presented below. Use this information however you'd like, but <a href="http://ucflow.blogspot.com/2011/05/throw-away-your-8-peak-beadsnow.html" target="_blank">use it with caution</a>. <br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEicG4LUNJPCE7ZIanEu8XR5e5z45IE4BylO8DBQh486iy3al1WQmdGKNSkwsxyG2DtGGBTNklfNMZBSrotHujTVk4RIuhs_w7IY_aP3eAUb2Zs_1-hwlfvFgSr3Ni0Cyw8dRt88Sq4RDerI/s1600/Avalon_8Peaks.png" imageanchor="1" style="display: inline !important; margin-left: auto; margin-right: auto; text-align: center;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEicG4LUNJPCE7ZIanEu8XR5e5z45IE4BylO8DBQh486iy3al1WQmdGKNSkwsxyG2DtGGBTNklfNMZBSrotHujTVk4RIuhs_w7IY_aP3eAUb2Zs_1-hwlfvFgSr3Ni0Cyw8dRt88Sq4RDerI/s320/Avalon_8Peaks.png" width="294" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">8-peak data collected by UCFlow during Demo</td></tr>
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So, we know it can sort, and we know it can resolve fluorescence adequately, but what about user-friendliness. Well, you've reached the best part! There's a single power button on the system; everything else is controlled through the software. There's a one-click startup sequence that primes the fluidics, and turns the stream on. There's a one-click, one bead alignement QC <u style="font-weight: bold;">AND</u> Drop-Delay assessment, and voila, you're ready-to-sort. There are a few details that occur behind the scenes here that are worth mentioning, but for the average user, they won't need to know any of this. It just works! So, what happens during the start up is a coarse alignment of the stream to the light collection pinhole. This is completely automated, and happens via pico-motor controllers on the x/y stage of the nozzle. The laser lines are fixed in place. Also, at this point, the system scans through the droplet frequency and amplitude, picks the optimal node, and optimizes the drop charge phase. After the coarse alignment, the QC/DropDelay beads are put on and the alignment is peaked. Lastly, it does a drop-delay test by sorting these same beads and counting the beads left in the waste trough at each adjustment in delay. In the waste trough is a small laser which excites the beads, and a fiber cable that carries any fluorescence to one of the systems PMTs. When the PMT fluorescence drops down to zero, the drop-delay is set. Again, all of this happens without any user intervention. Once set, the delay is monitored and adjusted as needed, and sorts are terminated in the case of a clog. The software, although a bit buggy yet, had a nice familiar feel taking a page from the MS Office suite and utilizing the Ribbon feature.</div>
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgq1TI4WAY5wsEkmGqLbD3JYKdxn1jkifsXOwa7YZsEMhwqcr_mZC_IWsceOQCq6fd829PfZkkWgTbd0CKbSuAV5VKhrpOG5_Y1uO7aIOJM31hBopaN3XOuVJL6HJcXW0Z6WachtQtD3qgV/s1600/software.jpeg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="194" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgq1TI4WAY5wsEkmGqLbD3JYKdxn1jkifsXOwa7YZsEMhwqcr_mZC_IWsceOQCq6fd829PfZkkWgTbd0CKbSuAV5VKhrpOG5_Y1uO7aIOJM31hBopaN3XOuVJL6HJcXW0Z6WachtQtD3qgV/s320/software.jpeg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Look and feel of Avalon Sorter Software</td></tr>
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The sample tube holder and collection receptacles are temperature controlled from 4C - 37C, and completely contained for excellent aerosol management. The sample tube holder has this really cool loading action. There's a front and rear position; the front position is where your sample is loaded, and the rear position is where the sample line is backflushed and rinsed, inside and out. You can see the loading and unloading action in the video below. Here, the Avalon covers are off for easy viewing.</div>
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Sort collection is equally unique. The system sorts 2-way, with a 5 postion tube holder (each side). So, you could load 10 collection tubes on the system, and once they fill the tubes in position 1, it'll automagically slide the next (empty) tubes into place. No more having to stop the sort, pull out full collection tubes, and put in new empty ones. The only complaint here is the lack of 4-way sorting and true, 96 well plate sorting. I say 'true' because there is actually an attachment that allows you to place two 8-well strips on the collection receptacle allowing you to sort 16 wells at a time. It certainly slows down your high-throughput sorting, but it works.</div>
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<tr><td class="tr-caption" style="text-align: center;">Looking into the sort chamber on the Avalon.<br />
5-position collection receptacle in foreground</td></tr>
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Final thoughts. The Avalon seems to capitalize on all the little things that so many companies got wrong in the past. Simplifying the design, minimizing the steps required to go from 'power-up' to 'ready-to-sort', and making a seemingly solid instrument, all add up to a very attractive package. To top things off, it comes at a bargain price, starting at just 99,950.00 US$ for a 1-laser, 2-color system. Sure, it won't replace the need for a 5-laser FACSAria III, but it can surely supplement your sorting needs by offloading those simple 2-4 color sorts clogging up your schedule. The only question then is, How soon can you scrounge up the funds?Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com3tag:blogger.com,1999:blog-8037707352330428788.post-77459501380857708212012-07-08T19:29:00.001-05:002013-06-20T12:03:42.274-05:00My 3-step approach to gating Annexin V data appropriatelyUsing flow cytometry to assess cell death, and more specifically, apoptosis, is an everyday occurrence in a flow core. Technically speaking, performing the assay couldn't be easier when utilizing numerous kits available from any number of vendors. It's typically set up in the format, add 'x' ul of reagent A to your cells, then 'x' ul of reagent B, and analyze. So, with such a simple assay, what could possibly go wrong?... Lots!<br />
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Let's assume there are no faults with setting up the instrument or parameter voltages and the like. Let's also assume the assay worked; that is, control cells looked relatively live, and treated cells looked nicely dead. The possible problem I've been coyly hinting at therefore, is in the analysis.<br />
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What's typically the first thing someone does when they analyze their flow data? They make a forward scatter (FS) versus side scatter (SS) plot and gate on the 'live' cells. Obviously, if you're trying to determine frequencies of live and dead cells, you wouldn't use a 'live' gate, you'd extend the gate lower on the FS parameter to include the dead cells. However, when you do so, you could be including small debris and bits of cells, and it's not exactly fair to count fragments of cells as events. So, herein lies the dilemma: You'd like to count whole cells, while drawing a light scatter gate that encompasses both live and dead/dying cells. Hence, <b>My 3-step approach to gating Annexin V data appropriately</b>.<br />
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<b>Step 1:</b> Display your Annexin V channel versus your Membrane Permeability Channel (for ease of typing, I'll assume we're using Annexin V FITC and PI) on the entire, ungated data set, and draw a region around the double negative (DN) fraction.<br />
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<b>Step 2:</b> Using the DN region from Step 1, gate a FS versus SS plot on these DN events, and d<span style="background-color: white;">raw a tight region around the subset of cells with low FS, now called Debris.</span><br />
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<b>Step 3:</b> Invert this Debris gate, creating a Not-Debris gate, and use this Not-Debris gate on the total population for your downstream Annexin V vs. PI display.<br />
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To see a screencast walkthrough of the gating process, watch the video below:<br />
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Let me break this down for you using an example. The entire point of the 3-step exercise is to properly define an area of the FS vs. SS plot that you'll call Debris, which will not be counted when generating your downstream frequencies. Depending on how much death you induce, and how toxic your treatment is, you could have a significant amount of Debris in your sample. It's important to note that you're not simply throwing away these events, you're just removing them from the downstream classification as Live (Annexin V neg, PI neg) Early Apoptotic (Annexin V pos, PI neg), Late Apoptotic (Annexin V pos, PI pos), or 'Necrotic' (Annexin V neg, PI pos). These debris events belong to none of these categories and therefore should not be included. The harm in including these debris events is that they usually end up in the Live fraction since they neither have a nucleus nor an intact membrane. Therefore, leaving them in inflates the Live fraction of cells, yielding potentially misleading results. The figure below shows the respective plots and regions.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhsuYiv9o7aGe1bC1HZrJnBToDjMu7sS3rNhsFbBc06tOTYmn60Q1soZJ0zLD7iQqLQznzRYMJ5Ex3q8isxLJFDDu4jYzZdvTTMdEybun5h-erCSl9wZgsHryI48RqU_DKYpPNmMDEqsEw3/s1600/AnnxPI_Gating.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="263" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhsuYiv9o7aGe1bC1HZrJnBToDjMu7sS3rNhsFbBc06tOTYmn60Q1soZJ0zLD7iQqLQznzRYMJ5Ex3q8isxLJFDDu4jYzZdvTTMdEybun5h-erCSl9wZgsHryI48RqU_DKYpPNmMDEqsEw3/s400/AnnxPI_Gating.png" width="400" /></a></div>
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Plot A shows a FS vs. SS plot of the full data set. Plot B shows the Annexin V and PI fluorescence on the entire data set with the double negative region drawn. In C, we are showing a plot of FS vs. SS gated on the DN cells. Here we can see we're left with two distinct populations. The larger population is the true Live cells (Annexin V neg, PI neg, and large), and the other population is the debris. I use a contour plot to show a nice outline of the region that should be drawn. This region is then applied to the top level FS vs. SS plot (as in D), and is inverted. This inverted Debris gate (i.e. Not-Debris) is then applied to the Annexin V vs. PI plot and quadrants are drawn to define the 4 populations present. </div>
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjpkj8nPkBvKarvncL0VaDq8LEaFpWcPquJxsYqO80red17Gn4LhUNPoYPf3-ffw3c15eHz5fHrnrXaRoDySRJP2hOijC4o6Dbv2kfTWBu2q6i4c2nZebW1rUh192eC-bGRn6MUxc4BT_F_/s1600/GatingApoptosis.png" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjpkj8nPkBvKarvncL0VaDq8LEaFpWcPquJxsYqO80red17Gn4LhUNPoYPf3-ffw3c15eHz5fHrnrXaRoDySRJP2hOijC4o6Dbv2kfTWBu2q6i4c2nZebW1rUh192eC-bGRn6MUxc4BT_F_/s200/GatingApoptosis.png" width="198" /></a>Not quite convinced yet? Well, I ran a similar sample on the ImageStreamX, gated in a similar way, but now, using the imaging power of the ISX, we're able to visually confirm what each of these populations is. To the left, we see the analogous plots and regions as prescribed in the 3-step method, and below we can see a representative pool of images from each of the regions. The panels labeled Debris and Live came from the DN gate drawn, and as in the flow cytometry data, the FS vs. SS plot (here presented as Bright Field Area and Side Scatter) separates into 2 distinct populations. A larger Live cell population and a smaller Debris population. Once the Debris is removed from the downstream classification, we are left with the Live, Early/Late Apoptotic, and Necrotic fractions. Of special note is the fact that we are not simply gating out small events, as there are small events that do in fact belong to the Necrotic or Apoptotic groups (see especially event #2269 in the Late Apoptotic panel). We're specifically gating out events that are small and have no fluorescence, which in this case, is a pretty good definition of debris. </div>
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Now obviously there are a whole bunch of caveats. This works really well for a homogeneously sized cell line, and perhaps less well for a mixed population. Also, as was briefly mentioned above, you'll need to still factor in that % Debris metric somehow. It could be your treatment makes either live cells or debris, and excluding the Debris from the Annexin V plot may show you have a healthy profile. However, it's important to track % Debris in addition to the other populations when comparing your conditions. If nothing else, though, you now have a reproducible method for gating out debris from your apoptosis analysis that could actually be explained properly in the methods section of your manuscript.</div>
<br />Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com6tag:blogger.com,1999:blog-8037707352330428788.post-83942065239147482212012-06-05T21:25:00.000-05:002012-06-05T21:26:47.796-05:00Fluorish vs. Chromocyte... READY, FIGHT!<br />
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One of the more difficult tasks people encounter when they start making multicolor flow cytometry panels is the process of finding antibodies, and knowing which fluorochromes to use. There's the primary task of finding the specific antibodies you want to use in a few fluorochrome options (hopefully) and figuring out which ones would work best on the instruments available. And then, there's a secondary task of optimizing the fluorochrome with the antibody in line with the adage: Dim fluors on abundant antigen, bright flours on sparse antigen. This secondary, but equally important task is a pretty advanced skill that is difficult to automate or build into an algorithm, although some companies, like Woodside Logic's <a href="http://www.woodsidelogic.com/CytoGenie/gettry-cytogenie.html" target="_blank">CytoGenie</a>, do a decent job of trying to handle this. However, in order to get to this second step, we need to first aggregate all the information from a lot of sources as to what's available. This step can be built into a utility, and it is precisely this step which I will discuss below. In particular, I'm going to focus on two offerings that have come online in recent years. One is the utility built into the <a href="http://www.chromocyte.com/" target="_blank">Chromocyte</a> web portal** under its 'calculate' tab, and the other is a utility developed by the parent of FlowJo called <a href="http://fluorish.com/" target="_blank">Fluorish</a>.</div>
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After playing around with both services for a bit, I'll highlight a few areas which can serve as a good comparison of the two. The areas I'll focus on are <b>Catalog</b>, <b>Hardware Integration</b>, and <b>Usability</b>.<br />
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<b><u>Catalog:</u> </b>First off, let's look at the catalog of antibodies available from the two sites. As expected, there's a lot of overlap, and they both have the catalogs of all the major vendors you'd expect to see. There were, however a few exceptions. For example, Fluorish.com doesn't have the catalog of EMD-Millipore, GeneTex, Cytognos, antibodies-online, Gen-probe Diaclone, and IMGENEX Corp - all of whom appear on the Chromocyte site. The Chromocyte site didn't have Miltenyi Biotec, and Sony/iCyt, both of which are present on Fluorish.com. Whether this translates into a use-case where you're unable to find a specific antibody you're looking for or not is debatable. I'd assume that nearly every antibody commercially available is likely to be on both sites, and if there is something not available, both sites prominently display a way to let them know so they can add it. However, it should be noted that the Chromocyte list seems to be outrageously exhaustive; allowing you to select antibody species from Alligator to Zebrafish, and everything in between. Another nice feature of both sites is the ability to select viability or other dyes, and fluorescent proteins so you can exclude those channels from the potential list of available fluorochromes. So far, we'll call this a truce, but wait...there's more. Fluorish.com, not only allows you to query all these commercial sources, but you can also add your own private stash to the database and allow those custom sources to be displayed in-line with the commercial sources. It just so happens that <a href="http://ucflow.blogspot.com/2012/01/introducing-cytometry-and-antibody.html" target="_blank">I'm part of a core facility</a> that also makes and conjugates antibodies, so this is a BIG DEAL for me. Catalog Winner: <b>FLUORISH.COM</b> </div>
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<tr><td class="tr-caption" style="text-align: center;">Fluorish's Panel Wizard showing Instruments in UCFlow Core.</td></tr>
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<u style="font-weight: bold;">Hardware Integration:</u> Next, I'm looking for the ability to integrate information regarding your facility's hardware. Again, both sites offer the opportunity to input and save the configuration of your specific instrument. Chromocyte uses a method where you select pre-defined filters from a list, whereas Fluorish has you input the cut-on and cut-off wavelength for each filter. Obviously, if your specific filter is not in the Chromocyte list, you're out of luck (although you could request your filter to be added). Using the Fluorish method allows you to precisely input your specific/custom filter, but it takes a bit of mental rigor (at least for my brain) as you try to calculate the cut-on and cut-off of a 595/14 BP filter. Fluorish, however, takes things one step further by allowing you to export your FACSDiVa configuration file from CS&T and drop it in, yielding a hands-free hardware configuration process. Both services have built in a layer of groups, which basically means I am set up on their site as a core facility, and other users can be part of my group, which gives them access to all the configurations I've already set up. As you can see, both utilities have done a very nice job allowing you to build panels specific to your hardware configuration, however with the abundance of FACSDiVa systems, the extra ability to drag and drop instrument configurations from CS&T into Fluorish puts it just over top of Chromocyte. Hardware Integration Winner (by a hair): <b>FLUORISH.COM</b><br />
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<u style="font-weight: bold;">Usability:</u> Lastly, but most importantly is Usability. This is such a huge topic, and one that's likely very personal to the individual user, that I'll simply report some of the things I really like or dislike about each of the services. When it comes time to pick which antibodies you will use, I really like the interface of Chromocyte where they lay everything out in a grid pattern. You can easily get a sense, at-a-glance, of how things might play out. If there are only a couple options under one of the columns, you'll know you should probably take care of that column (i.e. channel) first. The difficult part is that when you have a bunch of antibodies you're searching for and you take care of one of them (e.g. CD45), you'd expect the CD45 options in all the other channels would be greyed out or disappear. However, it allows you to accidentally select CD45 in multiple colors. Also, since the lists can sometimes be long, and if you choose one of the options that are off screen (i.e. an option you needed to scroll down to select), it would be nice if the column was greyed out or somehow highlighted to show you that channel is taken care of. <br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhdhHXFbDvfu-2CG-e35zMWikUrbWhdg25YsQC_cVjHpSA4G4KrHonhw6RxVIs9ozkF-fyIKQYeJbXB24oF5uJt1szw8rfdFcMgvvgXC3veFdQbx9rh9MaLuN9iUXV0Fzsb8WXX2P-CK4Zo/s1600/Chr_SelectingAb_Fluors.png" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="239" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhdhHXFbDvfu-2CG-e35zMWikUrbWhdg25YsQC_cVjHpSA4G4KrHonhw6RxVIs9ozkF-fyIKQYeJbXB24oF5uJt1szw8rfdFcMgvvgXC3veFdQbx9rh9MaLuN9iUXV0Fzsb8WXX2P-CK4Zo/s320/Chr_SelectingAb_Fluors.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Chromocyte's interface for picking conjugated antibodies</td></tr>
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Fluorish's answer to these problems is that it dims both antibody and fluorochrome options that remain once you choose your preference. Although Fluorish's interface isn't as pretty as Chromocytes, it's definitely more functional and easier to use. <br />
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A nice feature of Fluorish that's missing from Chromocyte is the ability to specify a specific clone of an antibody. So, if I know that I want to use RPA-T4 instead of OKT4, I can select that specific clone to refine my search. </div>
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An advantage of Chromocyte is the fact that it lives completely in the cloud. There's no software to download or upgrade, and so you're always using the latest version. With Fluorish, there are two sides of the equation. The first is Fluorish.com, which has limited functionality, but controls things like accounts, and core facility membership, etc... The second part is a program that you download called the Panel Wizard (currently on beta version 1.0b34). It works well, but completely fails at one crucial part of the equation... Mobile. Now Chromocyte is by no means optimized for mobile, but it at least works. Mobile is such a huge segment of my daily workflow, it's tough to forgive this omission on Fluorish's part. However, it's desktop application does work quite well, and with it's nice usability features discussed above, it more than makes up for that omission. Usability Winner: <b>FLUORISH.COM</b> </div>
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So, there you have it. In terms of a free utility to help you build panels for your multicolor flow cytometry needs, Fluorish.com appears to be a better option. **Of course, it must be said that Chromocyte has A LOT more to offer than its 'Calculate' tab. With its wide breadth of resources on flow cytometry techniques, services, news, and tutorials, Chromocyte is a one-stop-shop for the flow cytometry community. They've even <a href="http://www.chromocyte.com/educate/Training-Resources/Tips-for-Good-Flow-Cytometry-Experiments/UCFlow-10-Steps-to-a-Successful-Flow-Cytometry-Experiment" target="_blank">plugged one of my blog posts</a>, so you know they have good taste! Now we just need Fluorish and Chromocyte to get together and make a baby so we can have to best of both worlds. </div>Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com4tag:blogger.com,1999:blog-8037707352330428788.post-87664441097357029562012-03-27T15:55:00.001-05:002012-03-27T15:56:48.524-05:007 Habits of a Highly Successful Core FacilityI know, I know, another spin on the "7 Habits..." meme, but hey, reserve your judgement until you've read through them. This post is definitely geared towards core facilities, but I think you'll find this transcends to multiple service oriented businesses. Let me know if your facility is currently implementing any of these strategies, or ones that I've missed. And so, without further ado, I present the 7 Habits of Highly Successful Core Facilities, which was compiled after an exhaustive study of core facilities at the University of Chicago with an n of exactly 1 (i.e. us).<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi8i-Ii3635D8RpEx6MPU2AmI2FfQE7Bxq82rqFBgH0w0SEs5S1BnjeXlbUiYvMTdSDwYfXeN85afC1SZNgxsdVkpBmNdpvS-Le8ZwiFxVkzgFcBb-zdEioyGQWqegFm0d1SuSEhyphenhyphenO-CqF0/s1600/7habits-cover.png" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEi8i-Ii3635D8RpEx6MPU2AmI2FfQE7Bxq82rqFBgH0w0SEs5S1BnjeXlbUiYvMTdSDwYfXeN85afC1SZNgxsdVkpBmNdpvS-Le8ZwiFxVkzgFcBb-zdEioyGQWqegFm0d1SuSEhyphenhyphenO-CqF0/s320/7habits-cover.png" width="206" /></a>1. <b>Interact with OEMs</b> - I cannot stress enough the importance of having regular conversations with those companies who are making the products you are using. Many times labs will wait for a company to contact them with the latest and greatest products that have recently launched, however we've tried to take a proactive approach when it comes to new products and services. This invariably leads to demonstrations or evaluations, or even early access to new tech as a beta tester. In addition, you might just be able to voice your opinions so that future iterations of a product better suit your needs. <br />
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2. <b>Engage with your users regarding their scientific questions</b> - I've said multiple times before, people love to talk about their projects, so why not give them another opportunity to fine tune their story. I mean, you don't have anything better to do while sitting at the sorter, do you? The flip side of this is you may actually pick up on something and be able to suggest a technical improvement of the assay. There have been numerous times when someone is explaining to me what they're trying to do, and I've suggested using a different technology or approach, and it has worked out really well for them. For example, a user who I was training on a bench-top analyzer was complaining that it's impossible to differentiate between necrotic cells and late apoptotic cells definitely by flow cytometry. I simply suggested using our ImageStreamX instead (which also measures morphological parameters such as area of cytoplasm and darkfield scatter - a characteristic of necrotic cells) and the data supporting the hypothesis is now very convincing.<br />
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3. <b>Respond promptly to all inquiries</b> - Have you ever contacted a company only to have them ignore your attempts, or get back to you weeks later? How did that make you feel? Were you eager to do business with them again? Exactly! Whether it be by phone, email, IM, Facebook message/comment, or blog comment, it's imperative to follow up with people. Even if you don't have time to dig up an answer to someones question, at least be courteous enough to let them know you're working on the solution to their problem and that you'll get back to them is a day or two.<br />
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4. <b>Interact with people in many different networks (outside of your user base)</b> - It's easy to get into the habit of sending out an email blast to your user group. However, when you only do that, how will you increase your exposure and find new users? There are tons of opportunities to interact with both your current user base and potential user base, and the key is to pursue multiple avenues in order to cast a large net. Things like Facebook pages (e..g <a href="http://facebook.com/ucflow" target="_blank">UCFlow's FB page</a>, or Google+ pages (<a href="https://plus.google.com/u/0/b/100249365152403522736/100249365152403522736/posts" target="_blank">UCFlow's G+ page</a>) are great places to interact with people. It's also important to be both a contributor and consumer of information. Interact, comment, like, or plus things that other people are contributing to the social network, and promote groups that are doing things right. For example, I really enjoy the things that the Life Technologies group is doing on their Facebook page. I regularly read and interact with their content (<a href="http://facebook.com/cytometry" target="_blank">LifeTechnologies Flow Cytometry Facebook page</a>)<br />
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5. <b>Value quality over quantity</b> - At every point in your operations, it's imperative to emphasize the highest quality service. It's really about dotting the "i's" and crossing the "t's" ensuring each project is completed fully and to the user's satisfaction before moving on to the next project. With constant pressures to "do more with less" this is a difficult thing to follow through on, but I think it's important to give each project it's due diligence. Instead of sloppily or hastily completely as many tasks as possible, it'll pay off in the long run if you're focused on the task at hand and execute perfectly.<br />
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6. <b>Empower users (and faculty) to take ownership of the core facility equipment and services</b> - When you're users care just as much about the performance and health of the equipment and services as you do, you'll be in a very good place. One way of empowering them is by educating them on the implications of poor stewardship of the technology. "If you're running PI stained cells for cell cycle and you don't rinse properly, the next person who comes in with their PE, PerCP double stained fixed cells is going to end up with a ruined experiment." And, you wouldn't want that to happen to you, would you? Or, stressing the importance of filling up the sheath tank so that the pressure remains constant throughout your two hour run and you don't have fluctuations in the delay of laser #4. Also, when a user does have a problem on the instrument, don't simply walk over, fiddle with a few things and walk away. It would serve you much better to explain to the user exactly what you're doing, and how you came to the conclusions you did. Next time, they'll be able to do a bit of troubleshooting themselves. <br />
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7. <b>Be ready to pivot at a moments notice</b> - We in core facilities certainly believe our technology is the best, and will be around forever. But, that's rarely the case. Being able to assess the technology trends and stay in front of emerging/complimentary technology will allow you to stay relevant and grow. If there's no need for antiquated technology, you need to develop a plan to phase that instrument or service out and look to the next technology that will replace it. Holding on to the past, will leave you in the past.<br />
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So, there you have it. Why not make a quick assessment of your facility's habits and see how they stack up? Feel free to leave your thought below.<br />
<br />Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com0tag:blogger.com,1999:blog-8037707352330428788.post-50885485191652123072012-03-04T15:36:00.001-06:002012-03-04T15:36:16.768-06:00The Cytometrist's Mobile ToolboxIt's no secret technology continues to poke its nosy body into every part of our lives. But it's also surprising that we who work in the labs don't utilize it to its fullest potential. For example, up until recently, I would time my incubations by writing down on a scrap piece of paper what time (of day) the incubation was to end. I'd invariably lose the piece of paper and end up guessing how long it's been. This scenario plays itself over and over in various forms, which lead me to exclaim, there's got to be a better way.<br />
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So, whenever we're faced with a problem/question these days, where do we turn? Well, an app store, of course. Here, we'll outline a group of tools found on our phones/tablets/computers that can help working in a lab, especially in regards to flow cytometry applications, easier. <i>Quick Disclaimer: I use an Android phone, so most of my hands on experience comes from those apps. iOS apps were found by searching the app store for apps with similar features.</i><br />
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We call this our <b>Cytometrist's Mobile Toolbox</b><br />
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<tr><td class="tr-caption" style="text-align: center;">StopWatch&Timer+</td></tr>
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1. <b>Timer</b>. Running experiments always requires a good timer. Fortunately there are some really good apps for timing your incubations. My favorite one from the Android Market is called <a href="https://market.android.com/details?id=com.jupiterapps.stopwatch&feature=related_apps#?t=W251bGwsMSwyLDEwOSwiY29tLmp1cGl0ZXJhcHBzLnN0b3B3YXRjaCJd">StopWatch&Timer+</a> (sportstracklive.com). You can have many timers going at the same time with different alarms, and you can label them so you can easily go back to them for repeat incubations. You can also set timers up to 99:59:59, so you can cover your multi-day incubations. A comparable entry on the iOS App store is <a href="http://itunes.apple.com/us/app/timer+/id391564049?mt=8">Timer+</a>. It has a similar feature set except it only times up to 24 hours on a single timer.<br />
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2. <b>Calculator</b>. Sure, the stock calculator can handle most of the math you're going to run into day-to-day. However, the thing we probably do most is making dilutions, so it doesn't hurt to have a handy tool to do some quick dilutions and save you from making a stupid mistake. Most of the calculators you'll run into on the Android Market or the iOS App Store are built around chemistry applications, so you'll just have to train yourself to mentally substitute micrograms for micromolar as units. But other than that, they work well. There are some apps that have a group of tools (including some from BioLegend and LifeTechnologies, which I'll talk about below) which just makes it too crowded and requires lots of clicks (or touches, I should say), but on the Android platform, I use <a href="https://market.android.com/details?id=com.agilemedicine.agilescitools&feature=search_result#?t=W251bGwsMSwyLDEsImNvbS5hZ2lsZW1lZGljaW5lLmFnaWxlc2NpdG9vbHMiXQ..">AgileSciTools</a> with pretty good success. It actually does cell concentrations as well as stock dilutions (again using the mole base unit). A similar tool on iOS is <a href="http://itunes.apple.com/us/app/dailycalcs-science-calculator/id353223512?mt=8">DailyCalcs</a> (Invitrogen).<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgT3xETsj6qiNAZcn0e-JhrDoOLKqoL7e3yNvXAE0gaHfXYXYVjJzKnIzYJ1Ahyphenhyphen0PSU3fjs5uA2kYtPf7xxsYEIQ0-CvmK08tYZ-RGyjrWASNiQcOiCkY6Yg2TQKZQIy-j0tbX1FAJ1ehtt/s1600/dilutioincalc.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgT3xETsj6qiNAZcn0e-JhrDoOLKqoL7e3yNvXAE0gaHfXYXYVjJzKnIzYJ1Ahyphenhyphen0PSU3fjs5uA2kYtPf7xxsYEIQ0-CvmK08tYZ-RGyjrWASNiQcOiCkY6Yg2TQKZQIy-j0tbX1FAJ1ehtt/s200/dilutioincalc.jpg" width="133" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Dilution Calculator</td></tr>
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3. <b>Fluorescence Spectrum Resources</b>. Today, spectrum viewers are a dime/dozen, so it's no surprise that these are pretty easy to find. However, most of them exist as computer resource intensive web applications that aren't well-suited for viewing on a phone/tablet. In fact, there currently is no native android app that does a spectrum viewer like you'd expect from the various web site ones. Closest I've found is the <a href="https://market.android.com/details?id=air.com.invitrogen.cytometry&feature=more_from_developer#?t=W251bGwsMSwyLDEwMiwiYWlyLmNvbS5pbnZpdHJvZ2VuLmN5dG9tZXRyeSJd">Cytometry app</a> from Invitrogen. This doesn't have a spectra viewer, but it does at least list the ex/em of the various fluors they refer to. On the iOS platform, there is a <a href="http://itunes.apple.com/us/app/fluorescence-spectraviewer/id421031826?mt=8">full-fledged spectra viewer</a> from Invitrogen. So, if you're an Android user, you'll be better off using the various desktop apps. For a complete list of available desktop spectra viewers, you can visit the <a href="http://www.chromocyte.com/educate/Spectra-Viewers" target="_blank">Chromocyte web site</a>.<br />
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<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuFhwCJLWpTNI1L6_wb8vJhQdxlJIaMZcyDDebJIiX1T-a4ab8Sk_ilK6En7GwAEpoUiO2n2gGz7v1pZhpmUIzjZXMiZ1WG6l3l4qjdouwAHBXM-L59IQ2UqbAxpFZ6ewG5km0ax4F5zEs/s1600/CytometryApp.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjuFhwCJLWpTNI1L6_wb8vJhQdxlJIaMZcyDDebJIiX1T-a4ab8Sk_ilK6En7GwAEpoUiO2n2gGz7v1pZhpmUIzjZXMiZ1WG6l3l4qjdouwAHBXM-L59IQ2UqbAxpFZ6ewG5km0ax4F5zEs/s200/CytometryApp.jpg" width="112" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">LifeTechnologies Cytometry App</td></tr>
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4. <b>Antibody resource</b>. Sometimes when you're in the thick of it, you may want to get some info on a related antibody or reagent. Fortunately, this can be done with a few clicks on your favorite mobile device. BioLegend has a nice resource for looking up CD markers on both the <a href="https://market.android.com/details?id=com.biolegend.BLCD&feature=search_result#?t=W251bGwsMSwyLDEsImNvbS5iaW9sZWdlbmQuQkxDRCJd">Android</a> and <a href="http://itunes.apple.com/us/app/biolegend-cd-posters/id380861187?mt=8">iOS</a> platforms. Invitrogen's Cytometry app is also available on <a href="https://market.android.com/details?id=air.com.invitrogen.cytometry&feature=search_result#?t=W251bGwsMSwyLDEsImFpci5jb20uaW52aXRyb2dlbi5jeXRvbWV0cnkiXQ..">Android</a> and <a href="http://itunes.apple.com/us/app/cytometry/id494996119?mt=8">iOS</a>. <br />
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5. <b>Protocols</b>. Unsure of the protocol you're working on? Would you like to cross-reference another protocol, or even see some of the steps performed on video? Well, you're probably familiar with <a href="http://www.jove.com/">JoVE</a>, the online Journal of Visualized Experiments; a growing resource online. The only problem is that don't have an app yet (not sure why???). An alternative that does have an app is <a href="https://market.android.com/details?id=com.benchfly.app&feature=search_result#?t=W251bGwsMSwyLDEsImNvbS5iZW5jaGZseS5hcHAiXQ..">Benchfly</a> (Android only). However, as of this writing, there's not much flow cytometry related material there, although there are other related and useful videos ready-to-go. However, the place I start many times when specifically looking for video is YouTube. There are <a href="http://www.youtube.com/results?search_query=flow+cytometry&oq=flow+cytometry&aq=f&aqi=g10&aql=&gs_sm=3&gs_upl=1308l3580l0l3810l16l16l1l5l6l0l139l893l8.2l10l0" target="_blank">hundreds of videos</a> relating to many aspects of flow cytometry, and it is certainly available on any mobile device of choice. Also, the aforementioned BioLegend and LifeTechnologies apps have protocols built-in, using their products. Of special note is the LifeTechnologies app that actually has incubation timers inline with the protocol...pretty nice implementation. <br />
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Lastly, if you're looking for other tools, whether they be on the desktop of for your mobile device, you can consult the following links to some useful tools.<br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEju_IWJKv6ybyRgo51zRo_yg9rRNuDdcrcaVrvFANuWlUOYwvv7p6gbU4lZ4htQ57CKDvgLLbBbToRUXoN7ZdlWQFEHL9la7JKoCzAOtP5xKDZd1RX4hpAy4gQZzQfMxegTy0kys_bLG1Ze/s1600/biolegendtools.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="140" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEju_IWJKv6ybyRgo51zRo_yg9rRNuDdcrcaVrvFANuWlUOYwvv7p6gbU4lZ4htQ57CKDvgLLbBbToRUXoN7ZdlWQFEHL9la7JKoCzAOtP5xKDZd1RX4hpAy4gQZzQfMxegTy0kys_bLG1Ze/s200/biolegendtools.tiff" width="200" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">BioLegend Web Tools Page</td></tr>
</tbody></table>
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<a href="http://www.bdbiosciences.com/support/resources/flowcytometry/index.jsp#tools" target="_blank">BD tools</a>,<br />
<a href="http://www.lifetechnologies.com/us/en/home/communities-social/apps.html" target="_blank">LifeTechologies tools</a><br />
<a href="http://www.biolegend.com/webtoolstab" target="_blank">BioLegend Tools</a><br />
<a href="http://www.i-cyt.com/icyt_library.php" target="_blank">Sony i-Cyt tools</a><br />
<a href="http://www.chromocyte.com/" target="_blank">Chromocyte tools</a><br />
<a href="http://www.ebioscience.com/resources/fluorplan-spectra-viewer.htm" target="_blank">eBioscience tools</a><br />
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<br />
<br />
So, there you have it. Hopefully this will help you perform your experiments with greater accuracy (i.e. fewer mistakes) and efficiency. Do you have a favorite app you use to help expedite your experiments? Go ahead and leave your favs in the comments.<br />
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<br />Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com0tag:blogger.com,1999:blog-8037707352330428788.post-84723015624639474532012-02-14T21:41:00.000-06:002012-02-14T21:42:44.529-06:00Pinterest, the new place for social/scientific collaboration and discovery.<br />
A bunch of years ago a colleague of mine (some might know him as Marvin) and I had an idea that we wanted to create a repository of figures, images, and charts on our web site that we could point people to in order to show them what good cell cycle data looks like (for example) or to share a figure demonstrating what properly compensated and transformed data looks like. The thing that held us back were the technicalities of easily organizing and displaying these images. Over the years I've started and stopped various methods attempting to do this. Static web pages, a flickr pool, and shared volumes on a server, to name a few. But they were too cumbersome to use and therefore quickly faded as the mundane tasks of everyday life regained its stranglehold on my ambitions. <br />
<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjjKLl-TATI_I1TmziY9xm0FsOR_JAwM3EfObizyLN6Qk0RGB75h6nFLPZBYZjj-kkWnadBQ1FNchp5APhXMja4Vk-d4EW6NfqqGmE6UdYus2mk2J9EsSFM8uqfZjCaaZY3cwZSoSR-2_Vm/s1600/UCFlowFlickrPool.tiff" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="270" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjjKLl-TATI_I1TmziY9xm0FsOR_JAwM3EfObizyLN6Qk0RGB75h6nFLPZBYZjj-kkWnadBQ1FNchp5APhXMja4Vk-d4EW6NfqqGmE6UdYus2mk2J9EsSFM8uqfZjCaaZY3cwZSoSR-2_Vm/s320/UCFlowFlickrPool.tiff" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Dormant UCFlow Flickr Pool last updated in early 2009</td></tr>
</tbody></table>
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It took a bit of convincing, but I finally jumped on <a href="http://pinterest.com/">Pinterest</a>. If you've read any of the recent reports regarding the "average" Pinterest user, you could probably understand my hesitation. I mean, <a href="http://techcrunch.com/2012/02/11/pinterest-stats/">Techcrunch reported</a> that the fan base of Pinterest on FB is 97% female, and the Pinterest <a href="http://pinterest.com/about/">about page</a> describes its own ideal use case as, “People use pinboards to plan their weddings, decorate their homes, and organize their favorite recipes.” ...Not really the place you might find a science/technology guy. But it didn't take long for me to figure out how I could leverage the power of both the organizational structure of Pinterest (called pinboards), as well as the ease with which you can add images/figures to to the pinboards (that is, pin things to a board). Like many other social media type sites, you can follow people who pin interesting things. Most of the time, you can easily track back to the original source of the pinned image, but other times it leads to a dead end. This is a common complaint of the fledgling site. Some people have voiced concern over copyright and giving the original content provider his/her just credit. Hopefully some of these things will be ironed out as the platform develops. An <a href="http://www.stuckincustoms.com/2012/02/13/why-photographers-should-stop-complaining-about-copyright-and-embrace-pinterest/">encouraging perspective</a> on these copyright issues was recently outlined by world renowned photographer, Trey Ratcliff.<br />
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So, you can probably guess where I'm going here. Why not use Pinterest as sort of a repository of flow cytometry related images. Similar things have been done before, and probably with much more elegance, but I guarantee they have dedicated web professionals putting things together. Take, for example, a newer site that serves a similar purpose for microscopy imagery called <a href="http://www.cellimagelibrary.org/">The Cell: An Image Library</a>. It's a very professionally done site with lots of great content, but it likely requires a lot of upkeep and resources. Can a similar site of flow cytometry related images be created with minimal effort? We shall see.<br />
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The other thing to consider is this idea of peer reviewed images. 'The Cell' markets itself as a peer-reviewed repository of annotated images. I'm not interested in forming a review board, but since Pinterest allows multiple people to add to the same pinboard, you could envision a situation where people are adding things to a board, liking them, commenting on them, and using that as sort of the review process.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEikv0Wn0NN3_s2kkqy2ioz532Cg8DIRTA71WsblnW-hJJliV_ttTybmzb8BwH2knCZJj6R9DDDmC2gz52wgmXjSUBt_k_km-ErmAhT8Hfnc8NRcXOYRf87bsa6jPCHWBqQs1Kn3GBrg63xn/s1600/Pinboards.tiff" imageanchor="1" style="clear: left; float: left; margin-bottom: 1em; margin-right: 1em;"><img border="0" height="182" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEikv0Wn0NN3_s2kkqy2ioz532Cg8DIRTA71WsblnW-hJJliV_ttTybmzb8BwH2knCZJj6R9DDDmC2gz52wgmXjSUBt_k_km-ErmAhT8Hfnc8NRcXOYRf87bsa6jPCHWBqQs1Kn3GBrg63xn/s320/Pinboards.tiff" width="320" /></a>So if you're already a user of Pinterest, why not follow ucflow @ http://pinterest.com/ucflow/ and if you're interested in contributing to these pinboards, let me know. If you haven't had a chance to check out Pinterest, you should. Right now it's invite-only, but I can send you an invite if you'd like to seriously check it out. To the left, you can catch a glimpse at a few of the boards I've already set up. One board for hardware examples, one for figures of applications, and another generic board of flow stuff. It's a start, and could easily be branched into much more detail. For example, the applications board might get unruly and may need to be split up into application categories, such as Apoptosis Assays, etc... The only question is, will Pinterest succeed where other platforms have failed? Only time will tell.Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com0tag:blogger.com,1999:blog-8037707352330428788.post-66855074691145507112012-01-31T13:08:00.000-06:002012-01-31T13:08:02.006-06:00Introducing the Cytometry and Antibody Technology Facility (CAT)I can never tell if and when things like this become "official." It's not like administrators are going to put out a letter to the University announcing such things, so I thought I'd mark the occasion with a post about <b>it</b>. What is "it" you say? For years the Flow Cytometry Facility and Fitch Monoclonal Antibody Facility have been working together in a cooperative spirit. These efforts have mostly surrounded the creation of custom conjugated antibodies for use on our systems. They will now be fostered more deliberately in the formation of a new facility. And so it is with great pleasure that I announce that these two titans of technology shall become one - introducing the Cytometry and Antibody Technology Facility, CAT for short. Now, I must admit, I'm not crazy about the name either, but give it a chance, it'll grow on you. Just keep thinking to yourself, <a href="http://www.youtube.com/watch?feature=player_detailpage&v=RDjt9DRPhCI#t=47s">co-STAN-za</a>! Don't worry, UCFlow is not going away. The two subcores will retain their identities and remain pretty much intact, however we'll be able to leverage each other's resources to create some interesting new services for you. <br />
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; margin-left: 1em; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh-5sJ9eWZvpnaSevvwYN6WMAuxPB7vZuLKsWZxdU4z9aLkd9PKWDEH1qMrWkiC9U9MBou2N3hA6LUdfs6vEadwsASzcR1kwwTng868bI4opGw9_VIPZ8zFGJx_KAVtwMTLqg_kKUQ0h_FH/s1600/BirthofaMAB.jpg" imageanchor="1" style="clear: right; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="181" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEh-5sJ9eWZvpnaSevvwYN6WMAuxPB7vZuLKsWZxdU4z9aLkd9PKWDEH1qMrWkiC9U9MBou2N3hA6LUdfs6vEadwsASzcR1kwwTng868bI4opGw9_VIPZ8zFGJx_KAVtwMTLqg_kKUQ0h_FH/s320/BirthofaMAB.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">The birth of a mAb: Two cell divisions shortly after a subclone</td></tr>
</tbody></table>
If you've read entries here before, then you're probably familiar with UCFlow, so let me tell you a bit about our new partners, the Fitch Monoclonal Antibody Facility. For starters, the 'Fitch' part of the name is referring to cellular immunologist Frank W. Fitch, MD’53, SM’57, PhD’60 (University of Chicago), the Albert D. Lasker professor emeritus of pathology and the Ben May Institute, who joined Chicago’s faculty in 1957. As the institute’s third director, Fitch oversaw its growth into a collection of laboratories working in multiple areas of cancer research. A teacher and a mentor who encouraged students to think creatively while still abiding by scientific rigor, Fitch has also served as the editor in chief of the Journal of Immunology and as president of the American Association of Immunologists. He was a pioneer of antibody technology and was the founder of the Monoclonal Antibody Facility. Today, the facility's technical director is Ms. Carol McShan who has over 30 years of experience with tissue culture techniques, and novel monoclonal production. A major thrust of the facility over the years has been the production of novel monoclonal antibodies. This involves immunizing mice (or rats) with the specific antigen, screening the mice for a response, fusing the mouse's splenocytes with an immortalized fusion partner to create hybridomas, and finally subcloning and screening the hybridomas according to the end use of the antibody (flow cytometry, immunofluorescence, western blotting, etc...). In addition to novel monoclonal production, the facility also produces high-titer antibody supernatant, purified antibodies, and fluorescently coupled antibodies. A complete list of services can be found on their web site: <a href="http://fitchantibodies.uchicago.edu/">fitchantibodies.uchicago.edu</a>. <br />
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: left; margin-right: 1em; text-align: left;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj1VATxLw77yXs-GPjIxdMdaeYPOFM6KYLw7geG2Vd9mYSi9D2e2f8cSWO5Cp8-YbsyXZTOnlyLkQv8GfgTsPMHnGbMTXnEJAXYm3YI3R__RZuHweAA8aA4bhrG_z_Tzyzyq9p1-vBoajaq/s1600/BioreactorPic.jpg" imageanchor="1" style="clear: left; margin-bottom: 1em; margin-left: auto; margin-right: auto;"><img border="0" height="241" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEj1VATxLw77yXs-GPjIxdMdaeYPOFM6KYLw7geG2Vd9mYSi9D2e2f8cSWO5Cp8-YbsyXZTOnlyLkQv8GfgTsPMHnGbMTXnEJAXYm3YI3R__RZuHweAA8aA4bhrG_z_Tzyzyq9p1-vBoajaq/s320/BioreactorPic.jpg" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Hollow Fiber Bioreactors, utilized to produce high titer mAb.</td></tr>
</tbody></table>
Before we talk about what's in store for our joined future, let's discuss why I think this makes sense. I'm going to throw out a couple of economics terms to try and make a correlation to how things are done in the for-profit business world and then tie them back to what we're doing here. What we're really talking about here is a transition from lateral expansion to vertical expansion (or integration). My buddy wikipedia tells me that <a href="http://en.wikipedia.org/wiki/Lateral_expansion">lateral expansion</a> is the growth of a business enterprise through the acquisition of similar companies, in the hope of achieving economies of scale. Think of BD purchasing Cytopeia or Accuri. They gain scale due to the significant install base of these instruments. In a lot of ways this is pretty typical of successful companies. It can be a quick way to really expand your business and can lead to even greater growth. This lateral expansion sums up the past few years of the flow cytometry facility. We've added lots of pieces of equipment, grew our business, and expanded considerably. All of this expansion was pretty much of the same stuff - that is, cytometers. No, we weren't out 'buying up' other core facilities or things like that, but in our own way, we experienced a sort of lateral expansion. But there comes a point when you've pretty much maxed out this horizontally-directed growth and you need to pivot. So, what's the next logical step? You guessed it; vertical integration.<br />
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<a href="http://en.wikipedia.org/wiki/Vertical_integration">Vertical expansion</a> therefore, is the growth of a business enterprise through the acquisition of companies that produce the intermediate goods needed by the business or help market and distribute its product. This type of vertical pipeline integration is another very common strategy employed by companies. When BD purchased Pharmingen, that's exactly what was going on. If you are making the reagents that will be used on your instruments, you can control the entire package and make sure everything fits together nicely. This also works in reverse too. I'll remind you of the acquisition of Guava (and later, Amnis) by Millipore, or Invitrogen purchasing Applied Biosystems (becoming Life Technologies) and getting the supply of hardware to complement their reagents. This is sort of how I see the "merger" of UCFlow and the Fitch Monoclonal Facility. We can leverage the expertise in reagent development and production from the Monoclonal Facility so that our users have affordable and efficient access to commonly used reagents and will therefore be able to do more experiments with their limited funding (cf. more recharge revenue). Hopefully the synergy of expert reagent production and state-of-the-art technology will create a positive feedback loop for both sub-cores.<br />
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So what can you expect in the future? Our main focus at the start will be an expansion of the current Hybridoma Bank. Right now, the facility can produce purified antibody from 21 hybridomas including such favorites as anti-CD4 (GK1.5), anti-CD8 (2.43), the FC blocking antibody 2.4G2, anti-CD3 (2C11), NK1.1, CD19, anti-GFP, and many more. If we can get the hybridomas for an antibody, we can add it to our list. The next step to this is creating a plan to quickly couple these antibodies to a wide range of fluorochromes on-demand. We've spent some time with the <a href="http://www.innovabiosciences.com/antibody-labeling-kits.html">Lightning Link technology</a> from Innova Biosciences and it looks promising. This system allows for coupling to a good range of colors in as little as 20 minutes. We're still exploring many options, so if you have a favorite setup, let us know. We're excited about the prospects for this new phase of growth in the facility and will keep you posted about new developments as they happen.Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com5tag:blogger.com,1999:blog-8037707352330428788.post-73300339450808876642012-01-10T15:23:00.000-06:002012-01-10T15:24:30.027-06:00What to do with aging equipment: Upgrade or Replace? And a Mini MoFlo XDP Review.I started writing this post specifically to follow through on a <a href="http://ucflow.blogspot.com/2010/10/moflo-upgraded-to-xdp-plus-couple-new.html">comment I made</a> when talking about our upgrade of our aging MoFlo to the XDP platform (thanks for the reminder Carol). But then, I started thinking about all the equipment we've held onto and decided to upgrade and asked myself, was it really worth it? Before I answer that, let's lay out a bit of discussion on the matter, and then I'll finish up with my thoughts on our upgrade to the XDP.<br />
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When I think about equipment, I like to put things into 3 categories, namely Cutting Edge, Mid-Cycle, and End-of-life (EOL). I take all my equipment and shuffle them into these categories and move them around every so often as needed. This way, I can put things like service contracts, maintenance budgets, capital investment, and upgrades into perspective according to pre-determined criteria. For example, I'll stick instruments like our 4-laser Fortessa, or ImageStreamX into our Cutting Edge category. This means they probably won't require a huge maintenance budget since things aren't likely to break yet. However, they may need more personnel time because the applications performed on them are likely to be complex. I'll shuffle staff and training resources to those instruments. Mid-Cycle equipment are things like our 4 year old LSRIIs. Things are likely to start breaking and so they may eat up some service budget, but they are the workhorses and need to be running full-time. The applications are probably fairly routine, so they may not require as much custom tech time. Lastly, our EOL instruments are things like our ancient FACScan and FACSCantos, and it's these instruments on which we need to make decisions. Depending on the maintenance of these EOL'ed instruments, they may require varying amounts of service and since they may not be as desirable to use as the cutting edge cytometers, you'll need to determine how much money you're willing to invest to keep them limping along.<br />
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These EOL instruments can be a pretty decent consumer of budget and may or may not return all of their costs from recharge. It is with these instruments that we must decide; replace or upgrade (or I guess you could just let them die a slow death). You'll need to first determine if there is an upgrade path for your instrument. In the case of the MoFlo, this was a whole-hearted YES, thanks to the good folks at <a href="http://www.propel-labs.com/">Propel Labs</a>. Other instruments where this may be a possibility include FACScans and FACSCaliburs, which can be transformed into completely new instruments courtesy of <a href="http://www.cytekdev.com/">Cytek Development</a>. If there is not a path to upgrade, then the decision is an easy one. However, if you're looking into an upgrade, you'll need to weigh the costs against the benefits and definitely compare it to simply purchasing a brand new instrument. If you're going to shell out a bunch of money on an upgrade, it may make more sense to look into getting a new cytometer. Sure, you may have to settle with something a bit less powerful, but it'll be nice and shiny and (hopefully) problem-free for a few years.<br />
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So, let's put this all into practice with a retrospective look at our decision to upgrade our MoFlo. We had our MoFlo originally installed in 2000, and approaching 2008, it was definitely showing its age. Many of the buttons on the "rack" had fallen off, and it seemed like a waste of money to replace entire electronic bays on a rack to simply fix a button. In addition, parts to fix the MoFlo were somewhat scarce, and it looked like many of the components were approaching their demise. At the time, options for a new sorter were limited to the FACSAria, the inFlux (both from BD), the Reflection (from iCyt), and the MoFlo XDP (from Beckman Coulter). All of these instruments easily approached the $0.5Million mark, so buying a brand new sorter without an SIG or a generous donor was pretty much a long shot. Seeing as our MoFlo was still humming along just fine we decided to look into an upgrade. The goal going into this thought process was to have a sorter that would handle a lot of the cell line type sorts using GFP or other RFPs and perhaps a few phenotyping experiments. We did not have the expectation that it would rival our FACSAria and start performing multicolor phenotyping sorts as well as the Aria does. We also noted that our "GFP" sorts accounted for about 30% of all our sorts and guess what? We had 3 sorters; a perfect match. We were able to upgrade our MoFlo for about 1/3rd the cost of a new sorter and get a few more years of life out of it while we waited for the next big thing!<br />
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<b>MoFlo-XDP Mini Review</b>:<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg09HRTqs3gx9z7VyajbxXM_7FA7sWnknzsluTlawGdJ1sDoCZUhmVXqg9SyocShMwP4gmoSaxy1XonTIaLqGeJDfnxsO3vr_ZkQZP8NVUAgX2cOpXR6RwUVNYSmGcyiPTXMG-_PJhAXBHI/s1600/DSC_0252.JPG" imageanchor="1" style="clear: right; float: right; margin-bottom: 1em; margin-left: 1em;"><img border="0" height="212" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEg09HRTqs3gx9z7VyajbxXM_7FA7sWnknzsluTlawGdJ1sDoCZUhmVXqg9SyocShMwP4gmoSaxy1XonTIaLqGeJDfnxsO3vr_ZkQZP8NVUAgX2cOpXR6RwUVNYSmGcyiPTXMG-_PJhAXBHI/s320/DSC_0252.JPG" width="320" /></a>I can say that the XDP upgrade pretty much met our expectations. It handles most of our GFP/RFP sorts just fine, and is able to do a few more sorts on markers that are relatively bright. It by no means can resolve populations as cleanly as our Arias, but it does well enough for many things. The single best feature of the XDP is zero coincidence aborts. You may be thinking to yourself, well isn't the Aria marketed as having very low abort rates as well? It is, but when i say zero aborts, I really mean zero aborts, even when you have 30,000 - 40,000 events going through per second. The place this comes in handy are rare event sorts at high throughput rate. We can sort very rare populations and have a really good yield when compared to our Arias. What this really means, however, is that you have absolute control over your yield. If you need every single cell possible, you can run fast, have confidence that you'll be able to make a sort decision on every single cell, and using a yield sort mode, sort out every single cell. Sure, it won't be very pure, but at least you have them all and can decide to resort again if you need purity. Our specification for sorting yield is a 1% population with 70% yield using the purify sort mode (to achieve 98% purity or better). The max event rate able achieve this on the XDP is about 30,000 eps. The max rate able to achieve this on our Arias is slightly less (~22,000 eps). The touch screen is a bit annoying at first, but I've gotten use to it. The biggest problem with it is the implementation of the slider and up and down arrows. The slide is way too sensitive, and the up and down arrows are way too slow. This interface is used for adjusting things like frequency and amplitude and plate voltages. The new and "improved" Sort Master, dubbed Intellisort, works intermittently for us. It took a lot of playing around, but we can get it to hold onto a node pretty well these days, but for a while we completely ignored it. I still think this can be done way better, and apparently Intellisort II delivers, but I'm not going to hold my breath for that one. <br />
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So, am I happy with my decision? Absolutely! Would I do it again today? Not too sure. What it boils down to is, I spent a good chunk of change for a sorter that has 4 lasers and about 4 usable detectors at any given time. The need for the 4 lasers is pretty low, so I could get by with a 2-laser 4-color sorter and be able to do everything I'm currently doing on my 4-laser 10-color MoFlo-XDP. If I were given the option today, knowing what I planned to use the sorter for, I might check out the possibility of getting a brand new sorter that was stripped down to the basics for cell line transfection sorting. I'm thinking <a href="http://garvanflow.blogspot.com/2011/12/new-gear-and-new-year.html">something like this</a> perhaps might do the trick.Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com2tag:blogger.com,1999:blog-8037707352330428788.post-65154947402600753942011-12-19T15:54:00.000-06:002011-12-28T15:23:11.150-06:00Is Compensation really necessary?For some reason, it seems like the idea of compensation gets so much 'publicity'. Everyone is always talking about compensation and how difficult it is. New users of flow cytometry tend to think of this idea as something so complex that they end up stumbling on this one idea before they even get started. So, let's get one thing straight right off the bat; compensation is easy. In fact, I'd say compensation is ridiculously easy today, now that you really don't have to do anything. You just identify your single stained controls, and your software package uses that information to compensate your samples for you. The real difficulty in performing flow cytometry assays is panel design - determining which colors to use and coming up with a panel where you have the optimal fluorochrome coupled to each antibody to give you the best resolution of your populations. In fact, I'd go so far as to say that in some cases, compensation isn't even necessary.<br />
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Wha, Wha, Wha, What??? That's right ladies and gents - compensation isn't even necessary (<span style="font-size: xx-small;">in some cases</span>). And, I'm not just referring to the instances where you're using two colors that don't even overlap, I'm talking about straight-up FITC and PE off a 488nm laser. Now, before you stop reading and jump over to your Facebook feed let me just assure you that you first learned of the superfluous nature of compensation when you were about 5 years old. You see, analyzing flow cytometry data with or without compensation is nothing more than a simple "spot the difference" game you use to find in the back of the Highlights magazine while waiting to get your annual immunizations from the pediatrician. If you take a look at the figure below you may be able to recognize the left panel as the FMO (Fluorescence Minus One) control and the right panel as the sample. Spot the difference? Instead of seeing the sun missing on the left and then appearing on the right, let's just substitute a CD8-PE positive population for the sun. It doesn't really matter if the image is compensated, you're just comparing the differences between the two.<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgoumjlre9WRqZGdhxOHfMlpo75ZMovyxJqhiyxazr7WpF_MBnIbjknzTfs1-OcJkr_5aTPLYbTqU3kCpZ1hbYAfs4W2D5YWQPEwIw5JIS6UsoXK3yr2tGKmifQINlsid0J_1EfWK4n4nHf/s1600/SpotTheDifference.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="260" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgoumjlre9WRqZGdhxOHfMlpo75ZMovyxJqhiyxazr7WpF_MBnIbjknzTfs1-OcJkr_5aTPLYbTqU3kCpZ1hbYAfs4W2D5YWQPEwIw5JIS6UsoXK3yr2tGKmifQINlsid0J_1EfWK4n4nHf/s400/SpotTheDifference.png" width="400" /></a></div>
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Let's make the comparison a bit more directly. Here we have some flow cytometry data showing CD3 FITC and CD8 PE. Our goal is to determine what percentage of the cells are CD3+CD8+. Obviously, there's some overlap in the emission of the FITC fluorescence into the PE channel when run on a standard 488nm laser system with typical filters. If I were to hand you this data set and pose the question of "What's the % double positive," you could employ the same strategy used above in the spot the difference cartoon without knowing a thing about compensation. The top two plots below are the FMO controls (in this case, stained with CD3 FITC, but not stained with anything in the PE channel), and the bottom plots are the fully stained sample. In addition, the left column of plots were compensated using the FlowJo Compensation Wizard, and the right column of plots are uncompensated. Were you able to "spot the difference"? If you take a look at the results, you'll see that either way we come up with the same answer. So what's the point of compensating?<br />
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<a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiZB_wvzbPksLIhkZ1aUYPl1_BqwsPxKrQMJmgU28mML4YhYzdMcOeud4wjtuUKu97W_BUo13Q8nNopxvq2sjg344txAOoyl3Dc5VGv7d2rZ0z6DCDaGRRCGnUuZ984jQzJaGBwJUgbQvbj/s1600/IsCompNeeded.png" imageanchor="1" style="margin-left: 1em; margin-right: 1em;"><img border="0" height="356" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiZB_wvzbPksLIhkZ1aUYPl1_BqwsPxKrQMJmgU28mML4YhYzdMcOeud4wjtuUKu97W_BUo13Q8nNopxvq2sjg344txAOoyl3Dc5VGv7d2rZ0z6DCDaGRRCGnUuZ984jQzJaGBwJUgbQvbj/s400/IsCompNeeded.png" width="400" /></a></div>
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As you can imagine, this is greatly simplifying the situation, and when you start adding more and more colors, you simply cannot create an n-dimensional plot that can easily be displayed on a two-dimensional screen. This could easily work for 2-color experiments - it could even work for 3-color experiments (maybe using a 3-D plot), but beyond that, you're going to have to do one of two things. 1. Bite the bullet and get on the compensation train, or 2. Abandon visual, subjective data display altogether and move to completely objective machine-driven data analysis. Compensation, much like display transformation is a visual aid used to help us make sense of our data, two parameters at a time. In our example above, we don't magically create more separation between the CD3+ CD8- and CD3+ CD8+ populations. The separation between them is the same, we're just visualizing that separation on the higher end of the log scale (when uncompensated) where things are compressed in one case, and on the lower end of the log scale (when compensated) where things spread. You didn't gain a thing. </div>
<br />Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com13tag:blogger.com,1999:blog-8037707352330428788.post-48899962861921709512011-12-12T23:00:00.000-06:002011-12-12T23:00:34.858-06:0010 Steps to a Successful Flow Cytometry ExperimentI've been doing a good amount of application development recently and have had to "practice what I've preached." Those of us in the flow cytometry world, especially those in core facilities, like to pontificate all the do's and don'ts of flow cytometry, but how many of us have (recently) struggled through all the intricacies of perfecting a staining assay. I must say, I was a bit cavalier when I first agreed to set some protocols up for an investigator. The staining protocols weren't anything novel or difficult, it's just that I personally had not done some of the assays in quite a while. As I was going through the process I thought, hey, this is not as trivial as one might think...and I've been doing this for a loooooong time. I could only imagine what someone who is brand new to flow cytometry as a technique must feel like when their PI suggests they use this technology to investigate their hypothesis. So, I can put forth my top 10 steps to a successful flow experiment with some conviction, because I have now walked in your shoes.<br />
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I really wanted to make this a top-10, but as hard as I tried, I could only pare things down to 11. So, without further adieu I present to you;<br />
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<b><strike>10</strike> 11 Steps to a Successful Flow Cytometry Experiment </b><br />
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1. <b><i>Read lots of protocols (not just the reagent manufacturer's protocol)</i>.</b> Let's face it. If you ask a dozen people how to make a peanut butter and jelly sandwich, you'll end up with 12 different recipes. The same goes for FCM protocols. Everyone finds a different part of the protocol worthy of emphasis. If you read a few of them, you can start to put the entire story together.<br />
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2. <i><b>Know which colors work best on your instrument.</b></i> This is probably a bigger deal when you're using a core facility with a few different platforms. Let me tell you firsthand, no two cytometers are alike in their capabilities, not even two of the same model of cytometer. If you're lucky enough to have a flow cytometry core with knowledgable staff, make sure to ask them what their favorite 4, or 5, or 6-color panel is. They should also be able to tell you what the limitations of certain colors on a given instrument may be.<br />
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3. <i style="font-weight: bold;">When designing your panel, look for max brightness with min spillover.</i> Ok, let's say you know what sort of antibodies you want to run, and you know what's available, as far as hardware goes, at your institution. Now comes the fun part. You have a list of antibodies, and a list of fluorochromes - how do you match them up? You've probably heard the old adage, put your dim fluorochromes on the antibody that targets abundant antigen, and your bright fluorochromes on antibodies against sparse antigen. In addition to that you want to minimize spillover - fluorescence from probes that are excited by the same laser and whose emission overlaps. Spillover = Background, and Background = Diminished resolution. This takes some effort and a bit of know-how, so consult your friendly flow guru for help, or try out some of the new utilities designed to help with this process (namely <a href="http://www.woodsidelogic.com/CytoGenie/gettry-cytogenie.html">CytoGenie</a> from Woodside Logic or <a href="http://fluorish.com/">Fluorish</a> from Treestar).<br />
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4. <i style="font-weight: bold;">Titrate your reagents.</i> What for? The manufacturer told me to use 5ul per test (usually 10^6 cells in 100ul of volume). Without jumping on the conspiracy theory bandwagon that reagent manufacturers tell you to use too much antibody so that you'll waste your antibody and have to buy more, I will say that I've found more times than not that the manufacturers suggested dilution is too concentrated. If you want to see why you should titrate your antibodies, check out the figure below. If you want to see how to titrate your antibodies, click on over to this <a href="http://ucflow.blogspot.com/2009/06/antibody-titrations.html">prior entry to the UCFlow Blog</a>. <br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjVO7xTdSemA8k7qv4puwiq_IzxMJmDR799plgkUYBxwLuFCrb7QZrcwMgbywPJwZ5egNh_YSG7t_AyTJPh8_FXN-PXcUU6vCL6jGf5kBQeAWavg-Rhjx1nLWlrKb4KV_YaCjlzA6jB3ZsE/s1600/Titrate.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="160" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjVO7xTdSemA8k7qv4puwiq_IzxMJmDR799plgkUYBxwLuFCrb7QZrcwMgbywPJwZ5egNh_YSG7t_AyTJPh8_FXN-PXcUU6vCL6jGf5kBQeAWavg-Rhjx1nLWlrKb4KV_YaCjlzA6jB3ZsE/s320/Titrate.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><div style="text-align: justify;">
CD4 staining of fixed human PBMCs at the properly </div>
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titrated concentration (Left) and the manufacturer's </div>
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recommended concentration (Right). </div>
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<table cellpadding="0" cellspacing="0" class="tr-caption-container" style="float: right; text-align: right;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhSsnD5K4EJ1o8XrRCTI22_hJxEhglOJekuF8Te_NGZVg5vlxXB1-qxQFRSP3HV43UGPXO4TWVNs16-CcLb6y3r6wP0G8-WpNySgTbuV91LH9UETmtQqdUwVvXcHVuPT71hjzRQME8vZ0LO/s1600/ThSubsetWorklist.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="200" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEhSsnD5K4EJ1o8XrRCTI22_hJxEhglOJekuF8Te_NGZVg5vlxXB1-qxQFRSP3HV43UGPXO4TWVNs16-CcLb6y3r6wP0G8-WpNySgTbuV91LH9UETmtQqdUwVvXcHVuPT71hjzRQME8vZ0LO/s200/ThSubsetWorklist.png" width="131" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Example Staining Worklist </td></tr>
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5. <i style="font-weight: bold;">Outline your plan of attack.</i> Make a detailed work list of your protocol. Generic protocols are good to help plan your experiment, but when it comes time to perform the steps of an assay, you really want a work list. As the name implies, this is a step-by-step recipe of how to execute the protocol. I usually include the step, duration, volume of reagent, temperature, etc... While you're performing your assay, take copious notes so you can fine-tune the protocol, adding more detail. The goal is to be able to hand this work list and the reagents to another user and they should have successful results. I like to do this in Excel and write in all the cell formulas so that I can type in how many samples I need to stain and have it automagically do all my dilutions for me. I also have a summary of the buffers needed and quantities at the bottom. See below as an example.<br />
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6. <i style="font-weight: bold;">Always use a Dead Cell Marker.</i> Dead cells can really screw up an analysis. I guarantee there is a color and assay compatible dead cell marker available for most every experiment you will do. There's no excuse not to use a dead cell marker, so please, please do it. It makes for a much nicer looking plot, and you really can't do good (dim) double positive enumeration without it.<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEia3FFDq1hyphenhyphenaWOu3cCuMPWVR3Krjdx9zqep9UDE_vyBFUSMeBxwknTYoEwAC6GH44SXNC6R7HNk8O1_YAvAZ4vLhyFqy03QL-v-qHCOFWaFAXplnQotRTW38X7hu2hb1xNkbmEpEBWeCXd1/s1600/DeadCellExample.png" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="158" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEia3FFDq1hyphenhyphenaWOu3cCuMPWVR3Krjdx9zqep9UDE_vyBFUSMeBxwknTYoEwAC6GH44SXNC6R7HNk8O1_YAvAZ4vLhyFqy03QL-v-qHCOFWaFAXplnQotRTW38X7hu2hb1xNkbmEpEBWeCXd1/s320/DeadCellExample.png" width="320" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;"><div style="text-align: justify;">
Two-parameter plot without using an upstream dead cell </div>
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marker (Left) and the same plot after removing dead </div>
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cells (Right). Note the diagonal population extending </div>
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out of the negative population (encircled with a region </div>
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in the left plot)</div>
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7. <i style="font-weight: bold;">Set up your FMO's as a separate experiment, not on your real samples.</i> I won't discuss the merits of using an FMO control (<b>F</b>luorescence <b>M</b>inus <b>O</b>ne), let's just assume you know that it's pretty much a necessity. What I will say is if you try and set up an FMO control on the day that you're using your precious sample, you're likely to either forget it, or omit it because you think you don't have enough cells. So, if possible, set up your FMO controls ahead of time on a different day so you can take your time getting everything set up properly. It'd be nice to include it every time, if you have enough sample.<br />
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8. M<i style="font-weight: bold;">ake compensation controls using beads.</i> I'm a huge advocate of using capture beads to set up compensation. It's really a no brainer. I've written about <a href="http://ucflow.blogspot.com/2010/11/compensation-is-infiltrating-my-dreams.html">this subject</a> before. Even if your single stained controls look fine on cells, I'd still use beads because they're always consistent.<br />
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9. <i style="font-weight: bold;">Acquire your samples nice and slow to achieve maximum resolution.</i> If you go through the trouble of perfecting your staining procedure, now's not the time to screw things up. On a hydrodynamically focused instrument you'll want to concentrate your sample and run it slow in order to keep a narrow core stream and achieve optimal resolution. If you're using another type of flow cell (such as a capillary a la Millipore or an acoustically focused system like the Attune) you should be more focused on increases in background due to insufficient washing rather than a wide sample core.<br />
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10. <b><i>Analyze your data a couple of different ways.</i></b> Even if I have a clear idea of how to go about the analysis, I'm frequently surprised at how many times I've changed axes or started backwards and found I liked the new way better than the old way. Backgating is one way to help identify a rare population all the way up through its ancestry. Make sure to take advantage of your Live cell channel as well as gating out aggregates and removing any time slices where there may have been a drift in fluorescence.<br />
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11. <i style="font-weight: bold;">QC your instrument and create an application specific QA protocol.</i> Science is not about 1-shot deals. If it's not reproducible, it's not real. In order to give you the best possible chance of getting reproducible data you'll want to minimize the error contributed by the instrument. Quality control and Quality assurance cannot be emphasized enough. By doing something as simple as running beads at your application-specific voltage settings you can ensure that the instrument is in the same state as it was the last time you acquired these samples. For this, I typically use one of the peaks (peak 4, actually) of the 8-peak bead set. After I have the samples acquired with the proper voltage settings, I run the beads, create target channels for the peaks and save it as a template. Next time, all I need to do is dial in the voltage to put the beads in the target. You'll also want to make an Acquisition template and probably an analysis template too.<br />
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Well, there you have it. Hopefully this will help you focus your attention on some key aspects of setting up a well-thought-out flow cytometry staining protocol. Of course, this merely scratches the surface of all the things you need to think about. Did I miss something major? Feel free to leave a comment with your #12, #13, and beyond.Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com13tag:blogger.com,1999:blog-8037707352330428788.post-54611307956113884542011-12-02T10:31:00.001-06:002011-12-06T21:45:41.941-06:00The Year of AcquisitionsLike most industries, the Flow Cytometry Industry appears to be shrinking, in that the number of players on the industry side of things is getting smaller. For many years, there were a few big players, namely Becton Dickinson and Beckman Coulter (who they themselves were products of mergers - BD+Ortho, and Beckman+Coulter). They made instruments and reagents and pretty much sold the whole package. Seeing the potential for others to capture some of the market share, we experienced a growth of smaller start-ups, either focusing on the hardware or the reagents. Companies like Cytomation (maker of the MoFlo), and Guava on the instrument side of things introduced some nice products and created some much needed buzz. A major impact of these companies was that it forced the major companies to invest in R&D and come out with more competitive products. On the antibody side of things, reagent-focused companies like eBioscience and Biolegend gained popularity. But, I think a real turning point happened when little-known Accuri Cytometers exploded on the scene with a low-cost, small footprint cytometer with capabilities similar to a FACSCalibur. They took a page from the Guava playbook and targeted individual labs instead of the typical cytometer purchaser - a core facility. Soon other companies were seeing the success of these platforms, and the much larger market outside of the core facility. Companies like Stratedigm, Life Technologies and iCyt started offering smaller sized, less expensive cytometers. It seemed like the cytometery industry - both on the instrument and reagent side - was expanding. This lead to competition and innovation. The old standby's like BD and Beckman Coulter were forced to come up with new and exciting products to maintain their market share. And then the recession hit.<br />
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So, what happens in a recession. Well, contrary to what you might think, many companies do just fine in a recession. Of course their growth may slow, but then they also tend to accumulate capital as well. In fact many companies wind up in a situation where they have lots of cash on hand and are sort of waiting to see what's going to happen. John Waggoner explains in a USA Today piece (http://www.usatoday.com/money/perfi/columnist/waggon/2011-05-05-cash-in-on-mergers-and-aquisitions_n.htm) that this past summer, it was estimated that companies in the S&P 500 stock index had a combined $940 Billion in cash. I postulate that the well-established cytometry companies were/are in a similar boat...but to a much lower degree.<br />
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Mr. Waggoner goes on to explain, companies with cash-on-hand basically have three things they're going to do with it.<br />
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1. They can reinvest in the company, hire more people, build more plants, funnel it into R&D, etc... However, with funding becoming more and more scarce, there's not enough demand in the market to warrant such reinvestment.<br />
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2. They can return money to their investors in the form of dividends. Some companies are doing this, but probably in moderation.<br />
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3. They can buy another company to position themselves for the recovery. Mergers and Acquisitions are a pretty huge business in recent years. In total, M&As are running at a $1.6 Trillion pace for 2011. A good chunk of this is happening in the healthcare sector. <br />
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Bingo. Herein lies the recent increase in mergers and acquisitions. For example, Accuri raises ~$30 Million to get their business going, BD sees the threat and buys them for $205 Million (not a bad ROI for the Accuri Investors). BD removes the threat, and clears the way for its new flagship, small footprint, easy-to-use cytometer, the FACSVerse. This works for reagent companies too. Affymetrix buys eBioscience, EMD-Millipore buys Guava and now Amnis, Life Technologies licenses the acoustic focusing technology to build the Attune, and on and on it goes. Even bigger name companies like Sony and Danaher are getting into the game. Sony purchased iCyt to see if it can get its foot into the biomedical research arena, and Danaher purchased Beckman Coulter for who knows what reason. At any rate, it seems like the industry is attempting to go back to the old days where you'd do all your shopping at one company. Buy your instrument, reagents, analysis software, and all the rest from one company. You'll end up having BD labs, Millipore Labs, Life Technology Labs and maybe even Beckman Coulter Labs. A necessity in the current environment, but I'm sure things will oscillate back to the innovative start-ups taking on the big-boys once again. So, who's next to be gobbled up? I'm sure companies like Stratedigm, Blue Ocean, and Cyntellect are hoping their phones will start ringing.Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com2tag:blogger.com,1999:blog-8037707352330428788.post-24206148210722287542011-11-21T19:41:00.001-06:002011-11-21T22:23:23.797-06:00Options for Flow Cytometry Training - FloCyte ReviewFlow Cytometry (FCM) isn't the easiest technique to learn. It actually takes quite a while to master both the hardware and software components to sample acquisition and data analysis - let alone the applications utilizing the aforementioned instrumentation. For many users of flow (in an academic setting) their first encounter with FCM is likely through a core facility, whereby they'll receive some instruction on how to operate an instrument and then how to analyze the data they collected. The type and quality of this training varies greatly. Some institutions I'm familiar with have multi-day courses with wet lab sessions and hands-on instrument time, while others attempt to provide a theoretical base and then do a bit of hand-holding for a few sessions. The success a user may achieve greatly depends on his or her resourcefulness and overall aptitude for technology. Some people pick it up quickly; others struggle for years. I will say that training users in a busy core facility is a huge drain of time and resources. In our core, for example we basically have an entire F.T.E. just providing training and consultation, so I'm sure that in smaller cores, where it's just one or two people, training has to be an even greater burden. The question then becomes, how are we to provide the necessary training and attention our users require with the limited time and personnel resources characteristic of a core facility?<div>
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There aren't too many options. Before I jump into an assessment of the FloCyte courses (which is the whole point of this post) let me briefly highlight other possibilities. FYI, I've personally attended all 3 types of training sessions and have viewed all the resources in #4. </div>
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1. The Annual Course in Flow Cytometry - This weeklong course alternates between Los Alamos National Labs (or the University of New Mexico) and Bowdoin College in Brunswick, ME. It is really geared towards users of the technology who already have a basic understanding of the technology. Also, it focuses on the applications of flow cytometry rather than operation of a flow cytometer, however numerous sections also delve into the hardware components. There's a pretty cool lab where you can assemble your very own (fairly crude) cytometer. The cost of the course is about $1800, which includes dorm-style accommodations and meals (transportation is not included). </div>
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2. Vendor-specific instrument/software training - Most vendors will provide training for their hardware and associated software. When you purchase an instrument, you might get some free training included with the purchase, but additional training is going to cost you. As you'd expect, the training is geared towards the operation of that vendor's hardware. If you were using multiple cytometers from different vendors, this obviously wouldn't be ideal, but if you were using a single platform it might be a good option. The vendor training will also include some of the basics of cytometry, but again, it will be skewed towards their instruments, their reagents, and their idea of the technology. It's also pretty expensive, sometimes as much as $2500 per person.</div>
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3. Training courses at meeting - Typically when you go to some of the bigger conferences they'll have some workshops on FCM. Certainly at the <a href="http://cytoconference.org/">CYTO</a> meetings you'll have the opportunity to attend training sessions on various topics. Also, some of the immunology focused scientific meetings will have some FCM training associated with them (for example, the <a href="http://autumnimmunology.org/">AIC</a> meeting in Chicago). Cost for this training is variable, however it's usually limited to conference attendees, so unless you were already planning to attend the conference, it might be really expensive. </div>
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4. Online utilities - There is quite a lot of information freely available on the web. You can certainly start at the <a href="http://www.cyto.purdue.edu/flowcyt/educate.htm">Purdue University Cytometry Laboratory</a> web site, where there are a bunch of powerpoint slides, movies, and resources freely available. In addition, companies such as <a href="http://www.bdbiosciences.com/support/resources/">Becton Dickinson</a>, <a href="http://www.invitrogen.com/site/us/en/home/support/Tutorials.html">Life Technologies</a>, and <a href="https://www.beckmancoulter.com/wsrportal/wsr/support/training-education/flow-cytometry-webinars-conferences/index.htm">Beckman Coulter</a> offer overviews of flow cytometry and flow cytometer technology. Note that the above links are linked directly to the company's training/support page with the intended materials. Although these online utilities are readily available and free, you lose the benefit of asking questions and interacting with people who can tailor the training to your specific needs. </div>
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So now, I'll walk you through my experience with the FloCyte Training course offered by <a href="http://www.flocyte.org/">FloCyte Services</a>. I attended the Comprehensive Training Course from 11/15/11 - 11/17/11 held at <a href="http://www.spherotech.com/">Spherotech</a>, Inc. I won't bother taking up space here to give you the rundown of the company and the mission of the training courses. You can read all about it <a href="http://www.flocyte.org/FRTP/FRTP_introduction.html">here</a>. However, I will note that I attended the Comprehensive training course, which is designed for novice users of flow. You can see the course curriculum <a href="http://www.flocyte.org/FRTP/Course%20Descriptions/Comprehensive_Training_Curriculum.html">here</a>.</div>
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Day 1, as you'd expect, goes over the basic components of flow cytometry. This is done is a pretty common fashion, and anyone who's gone through the powerpoint slides on the Purdue University Cytometry Laboratory web site will recognize the format. 4-components, Fluidics, Optics, Electronics, and Data Analysis. All the standard material you'd expect to be here is here. There was however at least one pretty critical omission - multi-laser systems, laser delays, and how fluorescence emission is spatially separated. I know this was briefly mentioned during one of the sections, but there was no figure, no reiteration of how it's possible to look at two colors with the exact same emission simultaneously because they're excited by spatially separated laser beams (e.g. PECy7 and APCCy7). When we broke into small groups to take a look at some of the hardware, I spent most of the time explaining to my other group members how this works. They were very confused. The graphics used to talk about emission filtering where all systems like a FACScan or FACSCalibur, which don't have spatially separated beams, and all the light goes through the same "pinhole". Also on day 1, we finished up with a mathematical explanation of compensation, which went horribly wrong. The math is complicated and it's probably not something basic users need to understand in order to compensate their data correctly (or, should I say, let FlowJo compensate their data correctly). Lastly, there was no mention of 1 very critical component to flow cytometry, Quality Assurance and Quality Control. In all, the basics were handled just fine. I will say, though, that it seemed to move pretty slow. I think for the amount of information covered in that first day, it could've have been condensed into a half day. For example, I feel like the flow basics class given at UCFlow is comparable in it's scope but is completed in about 1.5 -2 hours. </div>
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Day 2 brought in a plethora of applications and tried to reinforce some of the concepts from day 1 while explaining how those concepts effect how you think about the applications. I think this way of presenting the information is really good. When we're talking about immunophenotyping, we're also talking about compensation, background due to fluorescence overlap, non-specific binding, etc... When we're talking about cell cycle, we're also looking at doublet discrimination, coincidence, sample core size, etc... Here we also start tackling the necessity of controls, including comp controls and the always popular FMO controls. My big issues with this section solely revolved around the figures. Many of the figures were at best poor representations of the idea being put forth and at worst blatantly misleading. This was especially noteworthy in regards to an explanation of biexponential display transformation. In another instance, the instructors were driving home the idea of how we are to never use quadrants to perform gating on our plots and the very next slide describing FMO controls was filled with quadrants used as gating. A bit contradictory.</div>
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Day 3 was all about stats and panel design. The stats part was very straight-forward and pretty easy to follow. The panel design section was good, and covered many of the issues that arise when trying to put together a multicolor panel. There was an introduction to a utility from Treestar called <a href="http://fluorish.com/">Fluorish</a> (which I'm not going to complain about because I like it) however there wasn't any real mention or demonstration of other available utilities like <a href="http://www.chromocyte.com/calculate">Chromocyte</a> and <a href="http://www.woodsidelogic.com/CytoGenie/gettry-cytogenie.html">CytoGenie</a>. Also, we spent some time going through some data analysis strategies using FlowJo.</div>
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The cost for the 3-day Comprehensive course is $700. The beauty of the course is that it's brought to you (either your institution can host it, or it is hosted nearby) so you don't have to factor in airfare or hotel costs. But, you'll have to remember that you're getting a comprehensive theoretical overview of flow cytometry, you are not learning how to operate your specific cytometer. So, if you didn't have a core facility around to show you how to open up FACSDiVa and adjust voltages on your LSRII, you'd still be pretty clueless on how to run your first FCM experiment. Another positive about the training is that it is modular such that you can attend just days 1 and 2, or just 2 and 3, or even just day 3. That way if you have some basic knowledge already, you can skip day 1 and just attend days 2 and 3. Lastly, I'll mention that there are a bunch of other, more <a href="http://www.flocyte.org/FRTP/Course%20Descriptions/Advanced_Course_Curricula.html">advanced courses</a> available outside the comprehensive course, including a multicolor compensation course, a course on "phosflow" assays, and even clinical flow cytometry. </div>
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The instructors are well-respected flow cytometry professionals with years of experience under their belts. They presented most of the material in a clear and concise way. There was, at times, some confusion regarding what a figure was trying to describe, but this was due to the fact that the slides were recently re-done and the instructors were not 100% comfortable with them. I feel like I want to give them a pass on that, but then again, I did pay $700 on this course and expected a very polished delivery. All things considered, they did an excellent job.</div>
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I could see this working in a couple of ways. 1. You get some initial training on how to operate your cytometer from your core facility and then attend days 2 and 3 of the comprehensive course. 2. You could attend the entire comprehensive course and then go through the specific instrument training given by your core facility. 3. Get trained by your core, start running experiments, and then jump in on one of the advanced courses offered by FloCyte. If you're not fortunate enough to have the support of a core facility, then this makes the FloCyte courses even more attractive. Relying on them for the basic theoretical training, and then the instrument vendor for training on the actual equipment is probably your best bet. </div>Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com3tag:blogger.com,1999:blog-8037707352330428788.post-18731262195863035262011-11-11T14:56:00.001-06:002011-11-14T15:30:23.432-06:00Cytometer Service Contract or Self Insure: the Wal-Mart effect.Instrument maintenance and repair is typically not a huge factor when deciding on a piece of equipment to purchase. People are much more concerned with the practical things like how many lasers can I put on, how fast can I run my samples, or more simply, can it handle the applications I plan to run? Even after we have the instrument installed in the lab we're not really thinking about maintenance and repair because we're on the "full-warranty high." If something breaks, what does it matter? The company will come out the next day and repair it at no cost. Right about the halfway point through the warranty period the thought hits you - I'm going to have to start paying for service on this thing. Herein lies the dilemma.<br />
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Although there are many variations, in general there are two schools of thought here. The first involves some level of service agreement (full, partial, lasers only, instrument minus lasers, etc...) and the second is akin to an "insurance" plan. By the way, before I go on, I should state that I'm writing this from the standpoint of a private academic institution (namely the University of Chicago), however private companies, public institutions, or individuals may have a vastly different experience. Let me briefly explain these two systems of instrument maintenance.<br />
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Service agreements. About 6-8 months into your warranty period, a friendly company representative will contact you to try and sell you on a full service contract. This basically extends the type of service experienced during the warranty period. Labor and parts will be covered under the service contract costs you pay annually. Be sure to get a list of what are typically called 'consumable parts'. These items are parts that will not be covered under the service contract. Consumables are commodities that are intended to be used up quickly and therefore are not parts that could undergo some type of failure. It is this failure of a part that is covered by the service contract. Consumables can be expensive; sometimes as much as $1000 - $2000 for a single item that may only last 6-12 months. You'll need to be sure to add these costs to your total cost of ownership. Full service contracts are fantastic. You get rapid response times, an endless supply of new parts, and generally I find the quality of service is of a higher standard. The downside is the expense. You can plan on spending about 10% of the original purchase price yearly on a full service contract, which means that after 10 years, you'll have bought the instrument twice. You can also look into service contracts that cover only parts of the instrument, such as a 'lasers-only' contract. This may cover some of the major expenses that might hit, but some of the routine fluidics issues or electronics issues would still need to be paid out-of-pocket. Lastly, you don't need to rely solely on the Original Equipment Manufacturer (OEM) for service. In some cases, third party companies will either provide the service agreement (serve as a middle man between you and the OEM) or there are companies that can actually come out and fix some of your older generation instruments.<br />
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Insurance. If you pass on the service agreement route, either with the OEM or a 3rd party company, you'll need to carefully make a plan on how you will pay for problems that pop up. This can be done by including a line item on your budget and simply inserting the cost of the service contract. Then you'd need to pay for any repairs using those available funds. If you don't use all the funds then you have a surplus and possibly a way to do some upgrades or save it for a rainy day. If, however, you end up paying out-of-pocket more than you have put away as insurance then you could have some trouble with your institution. The insurance method also has some unintended consequences including the possibility that your service calls may be bumped to the bottom of the list if the OEM services customers on service contract first. Secondly, I've noticed that the field service engineers tend to do the minimum to get the instrument functional again. This is not to say they're lazy or anything, they're actually doing you a favor by not replacing non-essential parts, and performing the work quickly so the hourly labor charge is not too high. However, this sometimes leads to more frequent trips to a site to fix a related part that breaks shortly after the instrument was put back into service. <br />
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So, what do we do at UCFlow? Well, a hybrid, of course. If you can anticipate which instruments will likely have more problems over the years, then you can keep your instruments running for many years without hassel for a lot less money. Seems impossible, but here are a few tricks. Obviously, the first thing you're going to do is monitor performance very carefully during the warranty period. If odd things are happening monthly, or even quarterly, it may be a good idea to consider a service contract. If you can find out from current owners of the same model instrument whether they have many service calls, that might help make the decision. Also, if you or your lab is familiar with the innards of the cytometer and aren't afraid to do things like replace valves, regulators, or even lasers then you should be less likely to buy a service contract. Lastly, the more instruments you have, the more money you'll be wasting on service contracts. Let's say you have 6 instruments, and the service contract is $15,000 each ($90K total). It's unlikely that all 6 cytometers will have multiple issues in a given year, so let's say you have 2 instruments with major problems (multiple service calls with big ticket items totaling $30K). The other 4 run pretty smoothly, and maybe require another couple of service calls for minor issues ($15K). If you pay out-of-pocket then you'll basically be paying 50% of the cost of a full service contract. This might be a good year; some other years might not be so favorable. However, it's likely that many years you'll be under budget and a couple of years you might be over budget. <br />
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As an example, I'll share a few stories of my experience. We had multiple 1st generation FACSCantos that were breaking down monthly. We were actually paying more out-of-pocket than the cost of a full service contract, so we went ahead and put them on contract. This was a no brainer. We also had an old LSRII that, over the course of 6 years had not had a single service call placed on it. All we've had to do is perform the standard Preventative Maintenance (PM). We never had a contract on this instrument. After 6 years of spending nothing on this instrument, we had 2 lasers die at the same time, which required replacement at the cost of $50K. The service contract cost was $22K per year, so 6 years times $22K = $132K, and actual costs were $50K, a 62% savings. It is a situation like this that tells us to err on the side of NOT getting a service contract until an instrument proves to be unreliable. Once it is deemed unreliable we either place it under service contract, or get rid of it and find a more reliable alternative. It sometimes seems like a gamble, and if I only had 1 or 2 instruments, I'd likely have them on service contracts, but since I have the luxury of duplicate technology and the power of numbers, I'm able to take that gamble and the odds are usually in my favor. <br />
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By the way, of the 16 instruments we have in the lab, 3 are on service contract (only the aforementioned early generation FACSCanto-A). We're able to save money by having a high number of instruments. We can also negotiate better contracts if desired. Larger volumes typically lead to better prices per unit. This is what we call the Wal-Mart effect. If that's not your case, then you'll likely want to lean more towards the service contract route. <br />
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<br />Anonymoushttp://www.blogger.com/profile/07384241547190280663noreply@blogger.com5