Beckman Coulter Resurfaces - Attend a Roadshow for info

It's remarkable to think that, until recently, the newest offering from Beckman Coulter was the FC500 (not counting acquisitions, like the Cyan and MoFlo)! It seems as though that is changing, rapidly. Beckman Coulter has recently announced two new players in the flow cytometer world. First up is the 3-laser, 10-color analyzer, Gallios. It seems pretty nice. I haven't gotten my hands on it yet, so I cannot give you the full run-down on it. They've certainly fixed a few problems that are found in their previous instrument (Colinear beams, more efficient light collection, better electronics, etc.) and so I'm sure it will be a huge step up from an FC500. However, there is still one thing that bugs me. It's billed as a RUO (research use only) instrument yet it has a tube carousel with a barcode reader on it!!! Seriously, who, outside of a clinical lab, uses a barcode system for their tubes. I find these carousel type loading systems to be really slow. I'd much rather pop tubes on and off, start and stop, add more buffer on the fly whenever I want. Instead, now I'm forced to use a carousel, and let it run. Not too happy about that. I do appreciate, however, the attention provided to light scattering. Light scattering seems like an afterthought on flow cytometers, but the Gallios has a forward scatter detector that can be optimized for small or large particles by adjusting the range of low angle light scatter detected. A wide position setting (1 to 19 degrees off normal) allows you to focus on smaller objects (bacteria, for example), while a narrow position (1 to 8 degrees) is ideal for larger cells. Lastly, you can use an enhanced wide angle module to focus in on submicron particle resolution. The data from this looks very promising.

The other new kid on the block is the Astrios (not sure about all the astronomical names), Coulter's new high-end cell sorter. First thing to note is, as I'm told, this is not a replacement of the MoFlo-XDP. The XDP will still be offered and developed. For how long? Who knows. The Astrios looks phenomenal. Here are a few stats: 6-way sorting, 7 spatially separated lasers, up to 30 fluorescence parameters, 0.3% Coincidence at 80,000 cells per second (cells, NOT beads). Is this for real? Time will tell, but after I wipe the drool from my mouth, I hope to get a test drive of this bad boy.

Along with the instruments, they will soon be releasing what I consider to be one of the best data analysis software packages around. Not just flow cytometry software, any data analysis software from any data intensive technology. It's called Kaluza, and it makes analyzing large data sets so easy and fast. I'm a FlowJo man, but the way this processes huge data files like it's nothing could turn anyone into a switcher. You just need to check it out to see it.

Speaking of checking things out, if you're in the Chicago-land area on May 21st, you can get an up-close-and-personal look at some of this technology. It's called the Flow Cytometry Big Bang Road Show, and it's coming to Chicago on May 21st, from 9AM to 4:30PM. More info can be found on the flow-centric Beckman-Coulter web site: www.coulterflow.com Or you can just go ahead and register for it here.

What is MFI?

If you've read any papers with flow cytometry data in it, undoubtedly you've come across the abbreviation, MFI. Generically, people expand this to Mean Fluorescence Intensity, but ironically, you'd rarely use the actual Mean of the population. Basically what the MFI is suppose to measure is the shift in fluorescence intensity of a population of cells. In cases where the entire population stains with different levels of an antibody (like measuring expression level of antigen x), it would be appropriate to report relative MFI values based on some sort of control (unstained, isotype, FMO, etc...) to demonstrate an increase or decrease in expression of this marker (assuming that each sample was stained with saturating amounts of antibody, and all samples were run under the same conditions and instrument settings blah, blah, blah). So, if you wanted to make measurements like this, what statistics would you use? When you analyze your data in software (e.g. FlowJo) you are given options to calculate the Mean, Median, Mode, and Geometric Mean. I've included a link which explains these measures in terms of flow cytometry data pretty well, so i won't bother going through that here. But, I will give you the punchline. When in doubt, use Median Fluorescence Intensity. Mean is pretty much useless, it doesn't work too well on a log scale, and for non-normal distributions, it is easily affected by outliers. I don't mean to be so mean when talking about the mean, but hey, for flow data on a log scale, why bother (sorry, i couldn't resist with the 'mean' pun). If you feel you must use an arithmetic average on a log scale, use Geometric Mean.

Welcome back Calcium Flux!

Is it just me, or does there seem to be a resurgence of calcium flux assays being done these days. Back in the day (don't laugh, I've been around for a relatively long time) calcium flux was pretty much standard procedure for immunologists and cell biologists alike. However, for various reasons, it fell out of favor over the past few years. Now, it seems like every few days, someone comes to talk to me about doing some calcium flux. You're probably well aware of Calcium's role in pretty much any cell activity, and the 'rapid' influx of the ubiquitous cation upon activation; so for those assays where you need to determine whether some stimulus actually causes the cells to increase activity or if that surface protein tail you modified causes a lack of downstream activity, this may be the perfect assay.

The way this assay works basically involves a fluorochrome which undergoes some sort of structural confirmation change upon binding Calcium, resulting in either an increase/decrease in fluorescence or a change in fluorescence absorption/emission. So, if you load that dye into a cell sample, you can track this change in fluorescence over time and quantify the rate of flux (how fast calcium rushes in), peak calcium flux (how much calcium rushed in), or duration of flux (how long the flux lasted). Some examples of these dyes include the perennial favorites, Indo-1, Fluo-4, and Fura Red, as well as some multitasking dyes like Oregon Green, and Calcein. Of course Molecular Probes (Invitrogen) has a calcium sensitive indicator dye in pretty much any color you can imagine. In addition to the favorites, they have a group of dyes cleverly called Calcium Green, Yellow, Orange, and Crimson. They all have slight variations in their chemistry, but all do pretty much the same thing. Whenever dealing with dyes and fluorochromes, there is no better place to look than the Molecular Probes Handbook, especially if you want way too much technical information than you could every imagine. Here's the link to the calcium indicator dye section if you want more info. Chapter 19, if you're a seasoned veteran of the handbook. The one major caveat that I need to bring up right here and now is that Indo-1 requires a UV light source, and not just any UV light source, it's gotta be really into the UV spectrum. We used to have a 325nm HeCd source, which was perfect for Indo-1. The only bad thing was the laser would only last about 6 months, and then the CVs were way too wide to be useable. We played that game for a couple of years, but have finally given up (almost) on UV altogether. We have violet sources, but no UV sources. But, do not fear, there are tons of options in every color for fitting in a Calcium indicator into your flow panel. And for any flow aficionados reading this post, yes we've tried the lightwave 355nm quasi-CW solid state laser. They are way too expensive, and only lasted about 18 months for us...we went through 2 of them. Our latest covetous thoughts are pointing us towards a 375nm diode. Not useful for Indo-1, but possibly good enough for DAPI cell cycle. As always, if you want to do calcium flux assays, but don't know how to get started, feel free to contact us day or night, but we'll probably only respond during the day.

Amnis ImageStream S10... Keep your fingers crossed!

We submitted our Shared Instrumentation Grant for the Amnis ImageStream Analyzer on Monday, and now we wait. For those not familiar with the ImageStream, it basically works like a flow cytometer, but instead of just getting relative fluorescence intensity units of different fluorochromes, it images the cells in as many as 5 fluorescence channels as well as brightfield and darkfield. This allows you to do some pretty interesting things, like co-localization of fluorescence with subcellular structure as in Nuclear Translocation. Additionally, we've requested some optional equipment like more laser lines (405nm, 488nm, and 658nm), and an extended field of depth (EDF) optical configuration. The EDF is useful in situations where you have punctate staining patterns dispersed throughout the nucleus or cell, as in fluorescence in-situ hybridization assays (FISH) or nuclear foci counting, as in gamma H2AX staining post irradiation treatment. We feel this instrument will greatly enhance the research goals of our investigators. To illustrate this point, we supplied project descriptions from 17 Principle Investigators demonstrating exactly how they would use this instrument. The support from the faculty was really impressive. We submitted very strong projects as justification for the need for this instrument, which yields some level of confidence in actually being funded. So, wish us luck, and hopefully next spring we'll have an ImageStream on campus.

Luminex Vendors Wooing New Customers

Is it just me, or have you been inundated with vendors trying to get you to use their bead-based assay kits. The two big wigs in this field are BioRad and Millipore (listed alphabetically as to not show any preferential treatment on my part) and they really want your business. As you may know, the Flow Cytometry Facility has a BioPlex machine in the Kovler Facility which gets moderate use on a relatively regular basis. I've met with both companies' sales force and they are very interested in hosting some seminars to show their products' worth. Being an equal opportunity obliger, I agreed and so we will be hosting 2 seminars regarding multiplex analyte kits. The first one will be from BioRad on February 26th at noon from 10:30 to 11:30 in Cummings Room 119. It will focus mostly on signaling protein detection from cell lysates, but I'm sure the technology in general will be discussed. This one will have food too! The second one from Millipore will be held on March 12th at 9AM also in Cummings Room 119 (go figure). The Millipore people are also bringing in some kits for you to test out some samples on the 12th and 13th, so this is a great opportunity to try something for free. If you need any information on this, send an email to ucflow'at'gmail'dot'com

Aria to Aria II upgrade...Painful, but worth it

Well that took way longer than expected. A two and a half day estimate went into its 5th day last friday. If anything could have gone wrong, it did. The procedure was going really well for the first two days, and then came the software. Why is it that whenever a problem occurs it always involves software glitches? Needless to say, it was a very frustrating afternoon on Thursday when we were completely stuck until the software started working properly. But, after some hard work and perseverance we finally got things up and running. We were able to start sorting on Friday, and to tell the truth, once we started, it became clear why we went through the trouble in the first place. The Aria started up so smoothly, and ran without a hiccup. You really can just pop in the nozzle and bam, the droplets just fall right into position. If you've ever tried to put the old-style nozzles in an Aria I (or had to use the wretched gold plated nozzles), then you'll really appreciate how easy it is on the Aria II. We've been running mostly with the 85um tip and have come to really appreciate that size. The 70um tip is good for going fast, but if the sample prep is not perfect, and the sample size homogenous and small, you get small jumps in the side streams which can really affect purity and yield. The 85um tip provides just enough room to still go pretty fast, and it's able to handle some junk too. The other thing that is instantly evident once you walk into the room is just how quiet the instrument is. The Aria I sounded like a rocket was taking off constantly, but now it's so quiet it's almost a little erie. Lastly, the charge plates are really nice. 4-way sorting is not even an issue any longer. In fact, the voltage sometimes is a bit too high so we've had to lower it to avoid hitting the plates with the side streams. So far, we've been really impressed with the performance of the instrument. One thing we're eager to get a feel for is how easy it is to keep clean. The jury is still out on that one, but we'll keep a close eye on it for sure.

Aria to Aria II upgrade...Day 2

Getting closer! Much of the Fluidics Board is hooked up and ready to go, the internal fluidic lines have been swapped. Today we'll tackle the flow cell, sample chamber, and sort block, and hopefully later, software and alignment. It probably seems like nothing has been done yet, but let me tell you, lots has been done. Bill and Scott (BD Engineers) have filled a 3' x 3' x 1.5' box with parts from the Aria I. Which reminds me, we'll have to find a new home for some of these parts, and the rest of them, well looks like I'll have to dig through some old Make magazines and see what we can put together.