2.10. Mitochondrial Membrane Potential (Δψm) Assay Using Flow Cytometry

Δψ_m was analyzed using the cationic dye JC-1 according to the manufacturer's protocol. A JC-1 stock solution was prepared at 1 mg/mL in dimethyl sulfoxide (DMSO) and stored at −20°C until use. After MPP⁺ treatment, cells were collected by centrifugation and washed twice with cold PBS. The pellets were resuspended in 0.5 mL of PBS and incubated with 1 µM JC-1 for 30 min at room temperature, shielded from light. The fluorescence intensities at 530 nm (FL-1) and 590 nm (FL-2) of 10,000 cells were measured using a flow cytometer. Δψ_m was calculated as the ratio of red (FL-2) to green (FL-1) fluorescence.