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.

中文翻译：

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通过差异标记的 MHC 多聚体优化抗原特异性 T 细胞的检测。

定期使用多种荧光分子标记主要组织相容性复合体 (MHC) 多聚体，以检测抗原特异性 T 细胞。我们已经评估了荧光标记的选择可以影响 MHC 多聚体结合 T 细胞检测的方式，该小组在一个探索性熟练面板中评估了 16 个不同实验室对 MHC 多聚体结合 T 细胞的检测。我们发现多聚体试剂的染色指数 (SI) 为 T 细胞检测研究提供了与给定荧光染料值的最佳直接相关性。SI 取决于流式细胞仪设置和选择的抗体面板；因此，最佳荧光染料选择可能因实验室而异。最后，我们描述了一种评估检测通道性能并优化所选荧光分子的 SI 的策略。这种方法可以很容易地用于测试和优化与 MHC 多聚体染色相关的荧光检测，一般来说，用于基于抗体的稀有细胞群鉴定。© 2019 作者。Cytometry Part A由 Wiley Periodicals, Inc. 代表 International Society for Advancement of Cytometry 出版。