Fluorescence-activated cell sorting (FACS) permits specific biologic parameters of cellular populations to be quantified in a high-throughput fashion based on their unique fluorescent properties. Relative quantitation of mitochondrial-localized dyes in human cells using FACS analysis allows sensitive analysis of a variety of mitochondrial parameters including mitochondrial content, mitochondrial membrane potential, and matrix oxidant burden. Here, we describe protocols that utilize FACS analysis of human lymphoblastoid cell lines (LCL) for relative quantitation of mitochondrial-localized fluorescent dye intensity. The specific dyes described include MitoTracker Green FM to assess mitochondrial content, tetramethylrhodamine ethyl ester (TMRE) to assess mitochondrial membrane potential, and MitoSOX Red to assess mitochondrial matrix oxidant burden. Representative results of FACS-based mitochondrial analyses demonstrate the variability of these three basic mitochondrial parameters in LCLs from healthy individuals, as well as the sensitivity of applying FACS analysis of LCLs to study the effects of pharmacologic induction and scavenging of oxidant stress.
