Optimization in Detection of Antigen‐Specific T Cells Through Differentially Labeled MHC Multimers

A large variety of fluorescent molecules are used on a regular basis to tag major histocompatibility complex (MHC) multimers for detection of antigen‐specific T cells. We have evaluated the way in which the choice of fluorescent label can impact the detection of MHC multimer binding T cells in an exploratory proficiency panel where detection of MHC multimer binding T cells was assessed across 16 different laboratories. We found that the staining index (SI) of the multimer reagent provided the best direct correlation with the value of a given fluorochrome for T cell detection studies. The SI is dependent on flow cytometer settings and chosen antibody panel; hence, the optimal fluorochrome selection may differ from lab to lab. Consequently, we describe a strategy to evaluate performance of the detection channels and optimize the SI for selected fluorescent molecules. This approach can easily be used to test and optimize fluorescence detection in relation to MHC multimer staining and in general, for antibody‐based identification of rare cell populations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.