Monday, April 27, 2009

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: Or you can just go ahead and register for it here.

Friday, April 17, 2009

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.

Tuesday, April 14, 2009

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.