Flow Cytometry Analysis of Mitochondrial Function in Muscle Stem Cells

Mitochondrial metabolism plays a central role in regulating muscle stem cell (MuSC) quiescence, activation, and differentiation, with dynamic metabolic transitions accompanying changes in cell fate and function. Flow cytometry-based analysis of mitochondrial function provides a rapid and accessible approach to characterize metabolic states at the single-cell level. Here, we present a detailed protocol to analyze mitochondrial activity, particularly by assessing mitochondrial membrane potential and mitochondrial mass in freshly isolated MuSCs and myoblasts using the fluorescent dyes TMRE and MitoTracker Deep Red in cytometry. This method is compatible with antibody-based identification of MuSCs and includes controls using mitochondrial uncouplers to validate ΔΨm-dependent staining. This protocol enables reliable discrimination between quiescent, activated, and differentiating muscle cells and can be applied to study physiological and pathological conditions in different contexts, including injury, aging, and diseases.