2.5. Measurement of a Mitochondrial Mass

A mitochondrial mass was determined by using the nonyl acridine orange (NAO) probe, which specifically binds to cardiolipin, a specific phospholipid that is found almost exclusively in the inner mitochondrial membrane and is widely considered as a mitochondrial mass marker [3]. For flow cytometry assay, cells were treated as indicated above, detached and fixed with 70% ethanol, and stored until use at -20°C. Then, ethanol was removed by centrifugation, and cells were washed in PBS and stained with 10 μM NAO in 1 mL of PBS. After incubating for 10-20 min at room temperature in the dark, cells were washed in PBS for three times and submitted for flow analysis. Relative change in mean fluorescence for green fluorescence at 530 nm was calculated which reflects the mitochondrial mass.