Measurement of mitochondrial membrane potential (ΔΨm) (MMP)

MMP was measured by flow cytometry using JC-1 staining. JC-1 dye exhibits potential- dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (∼529 nm) to red (∼590 nm). Mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. Following incubation with EX-527 or sirtinol, cells were further incubated with JC-1 dye (final concentration 2 µM) for 15 minutes. After incubation cells were washed with phosphate buffered saline (PBS) and re-suspended in PBS followed by flow cytometry. Carbonyl cyanide m-cholorophenylhydrazone (CCCP) was used as a positive control.