Guidelines for Gating Flow Cytometry Data for Immunological Assays

“Gating” refers to the selection of successive subpopulations of cells for analysis in flow cytometry. It is usually performed manually, based on expert knowledge of cell characteristics. However, there can be considerable disagreement in how gates should be applied, even between individuals experienced in the field. While clinical software often automates gating, and some guidelines do exist (especially for clinical assays), there are no comprehensive guidelines across the various types of immunological assays performed 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.