Mitochondrial Membrane Potential Analysis

Mitochondrial membrane potential was measured using a BD MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. The procedural details were described in our previous publications [33 (link),34 (link)]. All dead and viable cells were harvested, washed with PBS, and incubated with a 1× binding buffer containing the MMP-sensitive fluorescent dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolyl carbo-cyanine iodide) for 15 min at 37 °C in the dark. The cells were then washed, and JC-1 fluorescence was measured in a flow cytometer with excitation at 488 nm and emission at 530 nm (FL1-H channel for green fluorescence) for the monomer and 580 nm (FL2-H channel for red fluorescence) for aggregates, with a FACSCalibur flow cytometer using Cell Quest Pro software (BD Biosciences, Franklin Lakes, NJ, USA). The mitochondrial depolarization was measured as a function of the decrease in the red/green fluorescence intensity ratio.