How To Prepare For A Flow Cytometry Experiment

You never forget your first experiment.

Usually, you remember all the things that went wrong that you need to correct for future experiments.

In flow cytometry, there are so many things that you need to remember to bring to the first experiment, that having a checklist make sense. This checklist is divided into three phases — before the experiment, at the instrument, and after acquisition — with steps listed for each to help you perform your first flow cytometry experiment with ease.

1. Preparation before the Flow Cytometry experiment.

Before embarking on the first flow experiment, there are several things that you should do to become comfortable with the experimental plan. The first is to understand the protocol that will be used to stain the cells.

  1. Meet with the flow cytometry staff. One of the first steps to take before planning your first experiment is to meet with the team that manages the flow cytometer you will be using. These people are a great reference on the ins and outs of flow cytometry experimental design, execution, and analysis. You will probably have had training on how to acquire data, but a one-on-one meeting to discuss your assay and get feedback is a great way to learn more before embarking on the experiment.
  2. Sample preparation. For those starting with a liquid sample (pond water, blood, suspension culture cells), it’s pretty easy to get to the single cell suspension. When working with solid tissues, dissociation techniques become critical and references like the Worthington Tissue Dissociation Guide are excellent. Of course, don’t forget to filter samples before running them through the flow cytometer, especially if you are planning a sorting experiment. Finally, make sure to take a look at your cells under a microscope — it is your best friend to assess the quality of your single cell suspension.
Sample preparation before starting a flow cytometry experiment
  1. Gather and validate reagents. Make sure you have everything you will need, and ideally the reagents have been tested already. It really is frustrating to start a new experiment with a new technique using a reagent that you don’t know will work. New members in a lab might start with staining a standard sample with standard reagents until you are comfortable with all steps of the process. This sample is the same one used as a reference control in the assay, meaning there is a great deal of experience on how it should perform. Don’t forget to know any special issues with reagents — for example, don’t leave tandem dyes out in the light or on the bench for any length of time, lest they degrade and become useless.
Validate the reagents used for your flow cytometry experiment

Figure from Derek Davies.

  1. Book time. Make sure that you add some extra time as you get used to using the software. If you met with the staff already, ask them for an idea on how long they think the acquisition will take. Of course, don’t forget to add time to perform the necessary cleaning at the end of your acquisition.

2. At the cytometer.

Now comes the big day — the first experiment.

As with everything you do for the first time, it will take longer than you expect. Patience and care to follow the protocol are essential for success. Take a deep breath and begin.

  1. Double-check the necessary controls. Flow cytometry experiments require a large number of controls for successful interpretation. These include:
    1. Compensation controls — for setting compensation
    2. Fluorescence Minus One (FMO) controls — for assisting in gating
    3. Biological controls — for addressing variation in the assay
    4. QC controls — usually beads that will be used to monitor the QC of the assay over time
    Forgetting a control, especially in the first experiments, makes the downstream analysis harder. These controls each play an important part in the analysis, and forgetting one can mean success or failure. So double-check the list.
  2. Count your cells. Making sure you have enough cells for your experiment is important. Nothing ruins the day like realizing you don’t have enough cells to do what is needed. There are a host of ways to count cells,
  3. Make sure there are enough cells for accurate flow cytometry results
  4. Make sure you have your cheat sheets. Let’s face it, the checklist you got when you learned how to run the analyzer is important. It will help guide you through the process of using the software (a new skill) and ensure you don’t forget an important step (like hitting the “Record” button to save your data). Refer to it early and often. Soon, it will be like riding a bike, but for now the cheat sheet is your training wheels to get going.
  5. Arrive on time, if not a bit early. First-time jitters are common, so getting to the instrument a bit early doesn’t hurt. Being late, however, does. If you are early, let the staff know and see if anyone has time to be ‘on-call’ should you have problems. Also, make sure you transport your samples from your lab to the flow cytometer in an appropriate manner. Coolers like these from Igloo are a good choice because they are top-loading, have a closing lid (that ‘locks’) and provide a secondary containment for your samples. They also protect the sample from light.
  6. Annotate your data. Get in the habit early of using and entering keywords for all your experiments. In many acquisition packages, you can add keywords at the experiment level, the sample level, and the tube level. Using keywords and making a habit of it is something that will be its own reward when you are analyzing your data hours before a deadline. There are many things you can do with properly annotated data in third party software as well, so make sure you take advantage of this from the beginning.
  7. Follow the protocols. Check for the recommendations for best voltage, and how to collect your data and export it for later analysis. Make sure you collect enough events. Some packages default to a very low number of events, so check to be sure you are collecting enough data. At this point it’s time to run the samples and collect the data until the last tube is completed.

3. After acquisition.

There — the easy part is done. The first experiment is completed and in the books. Your data is being exported and saved to the place you’ve been told to save the data. Now what?

  1. Clean the machine. Follow the protocols for cleaning the instrument before the next user. Being a good citizen helps make core facilities run smoother, causes fewer issues for the next user, and in general is a good thing to do. Did you know, for example, leaving bleach on the sample injection port (SIP) can kill the next user’s fluorescence?
Thoroughly clean the flow cytometer when finished with your experiment

So follow the cleaning protocols — your fellow users will thank you. The core staff will thank you.

  1. Double-check the data is exported. Make sure the data is properly stored and you will be able to access and analyze it later. Sometimes there are hiccups in exporting the data, so checking to make sure each sample was exported (and there is data there) will save you a trip back to the instrument to get the data off a second time.
  2. Breath deep and prepare for analysis. Staining and running the experiment on the flow cytometer is the easy part. The hard part — and in many cases the fun part — is about to begin. That is the analysis of the data. Here, you will encounter your own set of new problems: from learning the analysis software, fighting the urge to ‘tweak’ your compensation matrix, making sense of the data, putting the gates in the right place, and so on. We’ll save your first analysis for another time. For now, breath deep, make sure everything is cleaned up, and enjoy that feeling from a first experiment.

You did it. You have survived the first of many flow cytometry experiments. Flow cytometry is a very powerful tool and can answer many questions if the experiments are properly designed. Using this checklist will help you design and perform consistent experiments every time. There is a learning curve that takes a bit of time, patience, and practice, but soon you may be finding excuses to perform flow cytometry experiments and we will be here to help you with best practices.

To learn more about how to prepare for a flow cytometry experiment, and to get access to all of our advanced materials including 20 training videos, presentations, workbooks, and private group membership, get on the Flow Cytometry Mastery Class wait list.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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