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MitoTracker ® dyes are fluorescent compounds that allow cellular mitochondrial content to be measured semi‐quantitatively by flow cytometry and have been used extensively in immunology publications. However, the parameters commonly reported, mean or median fluorescence intensity and percentage of cells that are MitoTracker® “high”, can be influenced by variability in cytometer setup, dye stability, and operator subjectivity, making it difficult to compare data between experiments. Here, we describe a method to identify MitoTracker® “high” populations in an objective manner. When analyzing data, we first removed outliers using a pre‐specified threshold, determined the fluorescence intensity of the brightest and dimmest events to obtain the fluorescence range and then gated cells within the top 90% of this range. This strategy substantially reduced variability between technical replicates and produced consistent results when data were analyzed by different operators. Consistent with previous reports and other analysis strategies, this analysis method demonstrated that within an individual, CD4 +  T cells exhibit significantly higher mitochondrial mass than CD8 +  T cells. Objective gating increases the reliability and utility of data generated using MitoTracker® dyes. © 2018 The Authors.  Cytometry Part A  published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

A Reproducible, Objective Method Using MitoTracker® Fluorescent Dyes to Assess Mitochondrial Mass in T Cells by Flow Cytometry