Bench philosophy: Eleven Flow Cytometry tips

Do’s and Don’ts of Flow Cytometry
by Ryan Duggan, Labtimes 06/2012

Though modern flow cytometers are becoming more and more user friendly and may even be operated by non-experts, flow cytometry is still a tricky business. Ryan Duggan, Technical Director of the Flow Cytometry Core Facility at the University of Chicago (UCFlow) gives some advice.

I’ve been doing a good amount of application development recently and have had to “practice what I’ve preached”. Those of us in the flow cytometry world, especially those in core facilities, like to pontificate all the do’s and don’ts of flow cytometry but how many of us have (recently) struggled through all the intricacies of perfecting a staining assay? I must say, I was a bit cavalier when I first agreed to set some protocols up for an investigator. The staining protocols weren’t anything novel or difficult; it’s just that I personally had not done some of the assays in quite a while.

As I was going through the process I thought, hey, this is not as trivial as one might think...and I’ve been doing this for a long time. I could only imagine what someone, who is brand new to flow cytometry as a technique, must feel like when their PI suggests they use this technology to investigate their hypothesis. So, I can put forth my top ten steps to a successful flow experiment with some conviction because I have now walked in your shoes.

I really wanted to make this a top-10 but as hard as I tried, I could only pare things down to 11. So, without further adieu I present to you: ten (11) steps to a successful Flow Cytometry experiment.

Practicing what he preaches: Ryan Duggan.

  • Read lots of protocols (not just the reagent manufacturer’s protocol). Let’s face it. If you ask a dozen people how to make a peanut butter and jelly sandwich, you’ll end up with twelve different recipes. The same goes for FCM protocols. Everyone finds a different part of the protocol worthy of emphasis. If you read a few of them, you can start to put the entire story together.
  • Know which colours work best on your instrument. This is probably a bigger deal when you’re using a core facility with a few different platforms. Let me tell you firsthand, no two cytometers are alike in their capabilities, not even two of the same model of cytometer. If you’re lucky enough to have a flow cytometry core with knowledgeable staff, be sure to ask them what their favourite 4, or 5, or 6-colour panel is. They should also be able to tell you what the limitations of certain colours on a given instrument may be.
  • When designing your panel, look for max. brightness with min. spillover. Okay, let’s say you know what sort of antibodies you want to run and you know what’s available, as far as hardware goes, at your institution. Now comes the fun part. You have a list of antibodies and a list of fluorochromes – how do you match them up? You’ve probably heard the old adage, put your dim fluorochromes on the antibody that targets abundant antigen and your bright fluorochromes on antibodies against sparse antigen. In addition to that you want to minimise spillover – fluorescence from probes that are excited by the same laser and whose emission overlaps. Spillover = background and background = diminished resolution. This takes some effort and a bit of know-how, so consult your friendly flow guru for help, or try out some of the new utilities designed to help with this process (namely CytoGenie from Woodside Logic or Fluorish from Treestar).
  • Titrate your reagents. What for? The manufacturer told me to use 5 μl per test (usually 106 cells in 100 μl of volume). Without jumping on the conspiracy-theory bandwagon that reagent manufacturers tell you to use too much antibody, so that you’ll waste your antibody and have to buy more, I will say that I’ve found more times than not that the manufacturers suggested dilution is too concentrated.
  • Outline your plan of attack. Make a detailed work list of your protocol. Generic protocols are good to help plan your experiment but when it comes time to perform the steps of an assay, you really want a work list. As the name implies, this is a step-by-step recipe of how to execute the protocol. I usually include the step, duration, volume of reagent, temperature, etc... While you’re performing your assay, take copious notes so you can fine-tune the protocol, adding more detail. The goal is to be able to hand this work list and the reagents to another user and they should have successful results. I like to do this in Excel and write in all the cell formulas so that I can type in how many samples I need to stain and have it automatically do all my dilutions for me. I also have a summary of the buffers needed and quantities at the bottom.
  • Always use a dead cell marker. Dead cells can really screw up an analysis. I guarantee there is a colour-and-assay-compatible dead cell marker available for most every experiment you will do. There’s no excuse not to use a dead cell marker, so please, please do it. It makes for a much nicer looking plot and you really can’t do good (dim), double-positive enumeration without it.
  • Set up your FMOs as a separate experiment, not on your real samples. I won’t discuss the merits of using an FMO control (Fluorescence Minus One), let’s just assume you know that it’s pretty much a necessity. What I will say is if you try and set up an FMO control on the day that you’re using your precious sample, you’re likely to either forget it or omit it because you think you don’t have enough cells. So, if possible, set up your FMO controls ahead of time on a different day so you can take your time getting everything set up properly. It’d be nice to include it every time, if you have enough sample.
  • Make compensation controls using beads. I’m a huge advocate of using capture beads to set up compensation. It’s really a no-brainer. Even if your single-stained controls look fine on cells, I’d still use beads because they’re always consistent.
  • Acquire your samples nice and slow to achieve maximum resolution. If you go through the trouble of perfecting your staining procedure, now’s not the time to screw things up. On a hydrodynamically-focused instrument you’ll want to concentrate your sample and run it slow in order to keep a narrow core stream and achieve optimal resolution. If you’re using another type of flow cell (such as a capillary à la Millipore or an acoustically-focussed system like the Attune) you should be more focussed on increases in background due to insufficient washing rather than a wide sample core.
  • Analyse your data a couple of different ways. Even if I have a clear idea of how to go about the analysis, I’m frequently surprised at how many times I’ve changed axes or started backwards and found I liked the new way better than the old way. Backgating is one way to help identify a rare population all the way up through its ancestry. Make sure you take advantage of your Live cell channel as well as gating out aggregates and removing any time slices where there may have been a drift in fluorescence.
  • Quality control (QC) your instrument and create an application-specific quality assurance (QA) protocol. Science is not about one-shot deals. If it’s not reproducible, it’s not real. In order to give you the best possible chance of getting reproducible data, you’ll want to minimise the error contributed by the instrument. Quality control and quality assurance cannot be emphasised enough. By doing something as simple as running beads at your application-specific voltage settings you can ensure that the instrument is in the same state as it was the last time you acquired these samples. For this, I typically use one of the peaks (peak 4, actually) of the 8-peak bead set. After I have the samples, acquired with the proper voltage settings, I run the beads, create target channels for the peaks and save it as a template. Next time, all I need to do is dial in the voltage to put the beads in the target. You’ll also want to make an acquisition template and probably an analysis template, too.

  • Well, there you have it. Hopefully, this will help you focus your attention on some key aspects of setting up a well-thought-out, flow cytometry staining protocol. Of course, this merely scratches the surface of all the things you need to think about. Did I miss something major? Feel free to leave a comment.

    Last Changed: 10.11.2012