Guidelines for Gating Flow Cytometry Data for Immunological Assays

"Gating" refers to the selection of successive sub-populations of cells for analysis in flow cytometry. While automated analysis algorithms have seen a greatly increased use, there is still heavy reliance on manual gating, based on expert knowledge of cell characteristics. This can lead to variability in how gates are applied, even between individuals experienced in the field. For clinical assays, guidelines often exist, and software may automate the gating process. But there are no comprehensive guidelines across the various types of immunological assays performed in research settings using flow cytometry. Here, we attempt to provide such guidelines, focused on the most general and pervasive types of gates, why they are important, and what recommendations can be made regarding their use. We do so through the display of example data, collected by academic, government, and industry representatives. These guidelines should be of value to both novice and experienced flow cytometrists analyzing a wide variety of immunological assays.