Mitochondrial Membrane Potential Assay

Mitochondrial membrane depolarization was determined using tetramethylrhodamine methyl ester (TMRM, ThermoFisher Scientific, T668) according to the manufacturer’s protocol. TMRM is a cationic, cell-permeant, red-orange fluorescent dye that accumulates in polarized mitochondria, but it is released after their depolarization. Untreated or treated cells were harvested, centrifuged and resuspended in culture medium containing 50 nM TMRM, and then incubated at 37 °C for 30 min in the dark. Cells were washed twice with PBS and immediately analyzed using flow cytometry.