Long 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.  

Why I love Listservs:

• 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.  
• 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 Purdue Cytometry List. For years (over 20, now) the Purdue List (as it's commonly referred to) has allowed cytometry professionals to interact and network.
• Being an active Listserv participant also gains you exposure, which can lead to new opportunities.  

Why I wish Listservs would crawl up into a little ball and be subjected to a slow painful demise:

• 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.  
• "Out-of-office" replies...need I say more?
• 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.   
• 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.
• 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.
• Wikipedia tells me that LISTSERV was developed in 1986.  Nineteen Hundred and Eight Six!!!!!  Do you know how long ago that was?  It was the last time the Bears won the Superbowl (OUCH!).
• Do we really need to be shuffling around emails to 4000 people on a listserv?  Answer: No.

21st century tools for a 21st century technology

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.  SOCIAL NETWORKING.  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:

• Sharing rich media inline with text to create better communication of ideas.
• A better sense of interactivity of the group instead of a one-to-one interaction.
• Fantastically good search tools for finding exactly the information you need
• Using email notification settings, you have the ability to interact as much or as little as you'd like.
• 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.
• Strong sense of community - Via avatars and in-depth profiles, you're able to build better relationships with colleagues.
• Expand your interaction with people on the fringe since they're already using these networks for other (personal and professional) purposes.

So, what's out there?  

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 Google+ Cytometry Community (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!

Life Technologies Attune Cytometer... Deja Vu

It wasn't so long ago that I blogged my impressions 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.

Fluidics:

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).

Software:

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.

There is no noticeable differences in Optics or Electronic other than the introduction of a Blue/Red laser configured instrument, which is built as a 4-2 configuration.

Data:

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.

Instruments at UCFlow tested for the qNORM characterization

qNORM values for 5 channels on the respective instruments.  Error bars are calculated from 4 separate experiments.

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.

Final Thoughts:  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.

Propel 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

I recently had the opportunity to check out the highly anticipated Avalon cell sorter from little-known Propel Labs (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.

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 "Nozzle-gate" 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.

The Propel Labs Avalon Cell Sorter

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.

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.

Sorting test using 3-color staining of fixed/nowash Peripheral Blood.

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 use it with caution.

8-peak data collected by UCFlow during Demo

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 AND 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.

Look and feel of Avalon Sorter Software

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.

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.

Looking into the sort chamber on the Avalon.
5-position collection receptacle in foreground

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?

My 3-step approach to gating Annexin V data appropriately

Using 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!

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.

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, My 3-step approach to gating Annexin V data appropriately.

Step 1:  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.

Step 2:  Using the DN region from Step 1, gate a FS versus SS plot on these DN events, and draw a tight region around the subset of cells with low FS, now called Debris.

Step 3:  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.

To see a screencast walkthrough of the gating process, watch the video below:

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.

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. 

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.  

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.

Fluorish vs. Chromocyte... READY, FIGHT!

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 CytoGenie, 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 Chromocyte web portal** under its 'calculate' tab, and the other is a utility developed by the parent of FlowJo called Fluorish.

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 Catalog, Hardware Integration, and Usability.

Catalog:  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 I'm part of a core facility that also makes and conjugates antibodies, so this is a BIG DEAL for me.  Catalog Winner:  FLUORISH.COM 

Fluorish's Panel Wizard showing Instruments in UCFlow Core.

Hardware Integration:  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):  FLUORISH.COM

Usability:  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.

Chromocyte's interface for picking conjugated antibodies

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.

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.  

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:  FLUORISH.COM  

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 plugged one of my blog posts, 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. 

7 Habits of a Highly Successful Core Facility

I 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).

1.  Interact with OEMs - 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.

2.  Engage with your users regarding their scientific questions - 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.

3.  Respond promptly to all inquiries - 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.

4.  Interact with people in many different networks (outside of your user base) - 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 UCFlow's FB page, or Google+ pages (UCFlow's G+ page) 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 (LifeTechnologies Flow Cytometry Facebook page)

5.  Value quality over quantity - 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.

6.  Empower users (and faculty) to take ownership of the core facility equipment and services - 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.

7.  Be ready to pivot at a moments notice - 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.

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.

The Cytometrist's Mobile Toolbox

It'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.

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.  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.

We call this our Cytometrist's Mobile Toolbox

StopWatch&Timer+

1.  Timer.  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 StopWatch&Timer+ (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 Timer+.  It has a similar feature set except it only times up to 24 hours on a single timer.

2.  Calculator.  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 AgileSciTools 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 DailyCalcs (Invitrogen).

Dilution Calculator

3.  Fluorescence Spectrum Resources.  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 Cytometry app 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 full-fledged spectra viewer 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 Chromocyte web site.

LifeTechnologies Cytometry App

4.  Antibody resource.  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 Android and iOS platforms.  Invitrogen's Cytometry app is also available on Android and iOS.

5.  Protocols.  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 JoVE, 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 Benchfly (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 hundreds of videos 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.

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.

BioLegend Web Tools Page

BD tools,
LifeTechologies tools
BioLegend Tools
Sony i-Cyt tools
Chromocyte tools
eBioscience tools



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.

Pinterest, the new place for social/scientific collaboration and discovery.

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.

Dormant UCFlow Flickr Pool last updated in early 2009

It took a bit of convincing, but I finally jumped on Pinterest.  If you've read any of the recent reports regarding the "average" Pinterest user, you could probably understand my hesitation.  I mean, Techcrunch reported that the fan base of Pinterest on FB is 97% female, and the Pinterest about page 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 encouraging perspective on these copyright issues was recently outlined by world renowned photographer, Trey Ratcliff.

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 The Cell: An Image Library.  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.

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.

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.

Introducing 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 it.  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, co-STAN-za!  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.

The birth of a mAb:  Two cell divisions shortly after a subclone

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:  fitchantibodies.uchicago.edu.

Hollow Fiber Bioreactors, utilized to produce high titer mAb.

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 lateral expansion 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.

Vertical expansion 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.

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 Lightning Link technology 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.

What 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 comment I made 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.

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.

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 Propel Labs.  Other instruments where this may be a possibility include FACScans and FACSCaliburs, which can be transformed into completely new instruments courtesy of Cytek Development.  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.

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!

MoFlo-XDP Mini Review:

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.

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 something like this perhaps might do the trick.