Measurement of mitochondrial mass and membrane potential

Adherent cells were stained in triplicate with 50 nM MitoTracker Green FM (Molecular Probes, Eugene, OR) in 1 × HBSS or 5 μg/mL JC-1 (Molecular Probes) in 1 × PBS for 15 min at 37°C. Cells were washed with 1 × HBSS, trypsinized, and washed twice more before analysis. For JC-1 controls, cells were treated with 10 μM carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP, Sigma) during staining. Using a BD LSRII flow cytometer (University of Rochester Medical Center Flow Cytometry Core, MitoTracker Green excitation 488 nm, emission 525 nm; JC-1 excitation 488 nm, green monomers detected at emission 525 nm and red aggregates at emission 610 nm), 10,000 events gated as singlets (FSC-A/SSC-A then SSC-H/SSC-W) were collected for analysis. FlowJo software v. 10 (FlowJo, Ashland, OR) was used to determine mean fluorescence intensity (MFI).