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.
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