FSC/SSC plot of PBMCs with gating for lymphocytes based on size and granularity. For primary cell analysis, such as for PBMC's, either lymphocytes, monocytes, granulocytes, or a combination of these are the initial gate on the FSC, SSC plot.įigure 1. If cell lines are being used, the FSC/SSC should show one main population of cells: this population should make up your initial gate. Knowing the size and makeup of your cells of interest is key to gating accurately. Larger, more complex cells will be higher in both parameters. The first step to isolating your cells of interest begins with forward scatter (FSC) and side scatter (SSC). Flow cytometry is often performed to look for immune markers and function, so we will focus on that here. Depending on your panel, gating can also allow you to look for differences between conditions within your cells of interest. Beginning with a broad gate of your cells of interest, gating narrows the population to cells of interest – cells inside the gate are included in further analysis, while cells outside the gate are excluded. Gating begins relatively the same regardless of the sample used, whether it is from primary cells or a cell line. Proper gating, along with smart panel-building, can make your data easier to interpret and more publication-ready. This includes general clean-up of the data, such as removing dead and dying cells or events consisting of multiple cells, as well as isolating your target cell population using their characteristic size, granularity, and expression of various cell markers. Gating is the process of selecting a subset of events from all events collected during a flow cytometry experiment for further analysis or data presentation. However, analysis software will allow for more comprehensive analysis of your FCS files and the ability to save your dot plots and histograms in a format that fits your needs. You’ve run your samples now what? Most flow cytometers allow some analysis while samples are running, as well as afterward. Written by Michelle Belmont, Scientist, MS at Proteintech Group.
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