A user's guide to multicolor flow cytometry panels for comprehensive immune profiling

Multicolor flow cytometry is an essential tool for studying the immune system in health and disease, allowing users to extract longitudinal multiparametric data from patient samples. The process is complicated by substantial variation in performance between each flow cytometry instrument, and analytical errors are therefore common. Here, we present an approach to overcome such limitations by applying a systematic workflow for pairing colors to markers optimized for the equipment intended to run the experiments. The workflow is exemplified by the design of four comprehensive flow cytometry panels for patients with hematological cancer. Methods for quality control, titration of antibodies, compensation, and staining of cells for obtaining optimal results are also addressed. Finally, to handle the large amounts of data generated by multicolor flow cytometry, unsupervised clustering techniques are used to identify significant subpopulations not detected by conventional sequential gating.

Automated summary

Multicolor flow cytometry is crucial for studying the immune system, but variations in instrument performance can lead to common analytical errors. By systematically pairing colors with markers and optimizing for specific equipment, designing comprehensive panels for flow cytometry experiments can help overcome these limitations. Unsupervised clustering techniques are then used to identify important subpopulations that may not be detected through traditional gating methods, allowing for better handling of the large amounts of data generated.