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.

Aria to Aria II upgrade...Day 1

The Flow Lab's legacy FACSAria is finally getting its fluidics upgrade today. The process takes about 3 days, so I'll give you some updates via photos. Day 1 was basically demo day, and the pic displayed here is the Fluidics area inside which has been totally ripped out. It looks a bit messy now, but the new system is much more stream-lined than the old. For those of you not familiar with the benefits of the fluidics upgrade, I'll give you a quick run-down and a bit of history. 1st the history. Sorters, for years have used a very simple process for driving fluidics; fill a tank with PBS, pressurize the tank at constant pressure, which causes the fluid to move through the system at a constant velocity, thereby giving a stable droplet break-off pattern. Enter BD and their quest to put a sorter on every researcher's desk. BD developed a fluidics system which was marketed as a "bench-top" sorter which could be run by anyone. Everything is automated, cleaning, sterilizing, running, monitoring, etc... In attempting to make this "easy-to-use" sorter, they created a service nightmare. There were so many valves and switches and pumps put in to do the simple job of moving PBS from a tank to a flow cell that at any given point, one of them was not working properly. Additionally, with all the valves and switches and automation came many small pieces of tubing and hangouts for bugs and bacteria. This created a situation where we had to jump through hoops trying to keep the sorter clean. Other sorters, like the MoFlo still had the classic fluidics system and, at least in our hands, had almost zero fluidics problems over about a 10 year period, and very few contamination issues with little effort. After a few years of complaints and retooling, BD releases the FACSAria II, and guess what? They got rid of a ton of valves, switches, hideouts, and once again went back to a simplified, robust fluidics system. It still has a few bells and whistles, but certainly not as grotesquely over-engineered as the Aria I. Will it withstand the onslaught of abuse only a core facility like ours can dole out? Only time will tell. Regardless, you'll hear about it here, so stay tuned.