Flow cytometry is a popular and powerful technique to investigate cellular signaling and function. Flow cytometry is quantitative and qualitative and capable of measuring intracellular and extracellular antigens within multiple cell types per sample. However, mistakes in experimental and panel design can result in false positive or false negative data and ruin the entire experiment. Here, we describe common pitfalls and important when working with flow cytometry.
Do: Understand the research question and hypothesis
Be clear on the cell types of interest, treatments, markers, and gating strategy before starting the experiment. Understand what question you are trying to answer and what information you need to gather, including whether your marker of interest is extracellular or intracellular.
Don’t: Underestimate the importance of panel design
Panel design is one of the most important steps for a successful flow cytometry experiment1. For multicolor panel design, balance the markers of interest over multiple lasers. Spread critical markers identifying different cell types out onto different lasers, if possible. For analyzing blood cell types, for example, the respective T and B cell markers CD3 and B220 can be put on one laser and CD4 or CD8 and CD19 can be used on a different laser. Highly expressed antigens should be paired with dim fluorophores, such as PerCP. Conversely, pair lowly expressed antigens or intracellular markers with bright fluorophores, such as PE. Include a dump channel, if necessary, which involves combining multiple negative markers into the same channel. Positive markers will be included in their own channels. Consider using an online panel design resource for complex multicolor panels2.
Do: Look up the lasers and filters of the instrumentation to be used
Understand which lasers and filters the cytometer you will be using has before designing your panel3. Many instruments have at least a blue (488 nm) and a red (633 nm) laser. Bandpass filters are the most common and allow a small range of wavelength to reach the detector. Ensure the excitation/emission spectra of your fluorophores are compatible with the lasers and bandpass filters of the flow cytometer to be used. If your panel only consists of 2 markers, it is unnecessary to run on an instrument that has 4 lasers and can run up to 25 colors.
Don’t: Forget that intracellular markers require permeabilization and fixation, which could affect extracellular staining
Intracellular staining is frequently performed to measure biomarkers of cellular signaling and function such as cytokines, transcription factors, phosphorylated proteins, cell cycle, and apoptosis. Intracellular staining often involves permeabilization with reagents such as Triton or Tween-20 and fixation with formaldehyde. If you will be staining cells with antibodies against both extracellular and intracellular markers, stain with extracellular markers before permeabilization and fixation steps to ensure that fixation doesn’t impair the staining of surface markers. Remember to save aliquots of unstained cells to have a control for unstained permeabilized and fixed cells.
Do: Use proper controls (single stains, FMO, biological)
Single stained controls can be prepared on the cells of interest or on commercially available compensation beads. If you don’t have very many samples and are limited on the number of cells for analysis, use compensation beads for controls. Fluorescence minus one (FMO) controls consist of cells or beads stained with all fluorophores minus one of them and should be included for complex multicolor flow panels. The purpose of FMO controls is to determine accurate gating. Always remember to use biological controls of untreated and/or vehicle-treated cells.
Don’t: Forget viability dyes
Dead cells bind nonspecifically to markers and will produce false positive data. Inexpensive viability markers include DNA-binding dyes such as DAPI, propidium iodide (PI), or 7-AAD. These dyes enter compromised cellular and nuclear membranes in dead cells but not live cells; however, they are not fixable. Fixable viability dyes include Live/Dead stains or Zombie dyes, among others, though fixable dyes are more expensive.
Do: Use proper cell concentrations
High cell concentrations reduce the accuracy of the analyzer. Dilute cells to 1 × 107/mL or less prior to running on the flow cytometer. The speed of acquisition is the event rate and can often be adjusted. For rare events, the maximum event rate should be set at 10,000 events per second, though lower event rates will increase accuracy of analysis.
Don’t: Clog the sip (use filters if isolating cells from tissues)
If you’re analyzing cells isolated from tissues, you may consider filtering your cells before running through the flow cytometer. If you can see clumps of cells or tissue in your samples before running, it’s imperative to filter large clumps out. If you think the flow cytometer is clogged, you can run a backflush and/or unclog protocol. Just remember to switch out your sample with a tube containing water before backflushing.
Do: Enrich rare cell types
If you’re interested in rare cell types, you can enrich rare cells or deplete unwanted cells using magnetic separation kits. This will save running time on the flow cytometer and will reduce the amount of data to be stored. More time should be allowed during the staining protocol if a magnetic separation step is to be included.
Don’t: Analyze junk events
Gate out erroneous events such as debris, cell doublets, dead cells or incorrect cell types based on surface markers of interest. Debris is smaller than cells based on FSC (forward scatter) vs SSC (side scatter). Doublets can be excluded based on FSC-H (height) vs FSC-A (area) or SSC-H vs SSC-A. Dead cells can be excluded with the addition of a viability dye.
Do: Backup your data
Flow cytometers are often shared among multiple people, laboratories or even departments. The common curtesy for any shared instrument or computer is to backup your data onto an external hard drive and/or cloud and remove data stored on shared computers. Always have multiple backups in case laptops or hard drives become lost or destroyed.
While flow cytometry initially involves a lot of time and effort to design and optimize multicolor panels, the effort is worthwhile as the same panels can be used for repeat experiments and the amount of data that can be collected from samples is tremendous.
References
1. Panel Design https://www.bdbiosciences.com/en-us/resources/panel-design.
2. Flow Cytometry Panel Design I FluoroFinder Tools FluoroFinder. https://fluorofinder.com/flow-cytometry-panel-design/.
3. Optics of a Flow Cytometer - US https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/flow-cytometry-basics/flow-cytometry-fundamentals/optics-flow-cytometer.html.
About the author: Bri Hoover, Ph.D. is a scientist with 12+ years of academic and pharmaceutical industry experience in discovery research & pre-clinical drug development. Bri received advanced education in biotechnology, hematology, and oncology from the University of California, Irvine.