Assaying Mitochondrial Function by Multiparametric Flow Cytometry

Flow cytometry has been a vital tool in cell biology for decades based on its versatile ability to detect and quantifiably measure both physical and chemical attributes of individual cells within a larger population. More recently, advances in flow cytometry have enabled nanoparticle detection. This is particularly applicable to mitochondria, which, as intracellular organelles have distinct subpopulations that can be evaluated based on differences in functional, physical, and chemical attributes, in a manner analogous to cells. This includes distinctions based on size, mitochondrial membrane potential (ΔΨ_m), chemical properties, and protein expression on the outer mitochondrial membrane in intact, functional organelles and internally in fixed samples. This method allows for multiparametric analysis of subpopulations of mitochondria, as well as collection for downstream analysis down to the level of a single organelle. The present protocol describes a framework for analysis and sorting mitochondria by flow cytometry, termed fluorescence activated mitochondrial sorting (FAMS), based on the separation of individual mitochondria belonging to subpopulations of interest using fluorescent dyes and antibody labeling.