Standard methods for JC-1 detection are not optimal. L1210 cells were first incubated with 1 μM valinomycin or DMSO (control) for 30 min at room temperature (RT) and then stained with 2.5 μM JC-1 for additional 15 min (RT). Valinomycin and mock-treated cell populations were analyzed either separately or mixed by the Gallios Flow Cytometer (Beckman-Coulter), using a 488 laser, following standard protocols provided by JC-1 manufacturers. Data were processed by FlowJo v7.6.4. FL2 (585/42)−FL1 (525/50) subtraction (compensation) are shown in percentages.