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A guide to gating in flow cytometry

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Flow cytometry analysis typically begins with creating gates to distinguish cells of interest. This process of gating can appear quite random to a flow cytometry novice but it is in fact the most important part of flow cytometry analysis.

So what gating methods do you need to know to confidently analyze your stained samples? This blog post will take you through the various gating strategies for effective flow cytometry analysis.

Learn as much as possible about your cells of interest

Your gating strategy is informed by what you know about your cells of interest. Therefore, before you begin your analysis, it is important to first find out as much as possible about the cells you are analyzing.

Things to determine are the relative expression levels of cell specific markers, the approximate size of the cells, and whether their size can be affected by experimental conditions. For example, the fixation and permeabilization processes during intracellular staining can alter cell size and granularity, resulting in modified forward and side scatter profiles (discussed below).

If you have no prior experience with your cells of interest, it is important to check the literature as a guide. Before beginning, you should also ensure that you include proper controls for accurate data analysis. Our recent webinar on flow cytometry controls provides information on what controls to include in your experiment.