Mitochondrial membrane potential

Mitochondrial membrane potential was evaluated using DiOC6, JC-1 and TMRM. HEK293 cells were seeded 2 days and synchronized by medium change 1 day prior to the measurement. For each condition, 1mio cells were added to six FACS tubes and washed with DPBS. JC-1 staining solution was prepared by mixing JC-1 stock (5mg/ml) with DMSO (1:3) and subsequent dilution in DMEM+10%FCS+1%P/S to a final concentration of 2.5μg/ml; cells were stained for 15min at 37°C in the dark. For staining cells with DiOC6 or TMRM, cells were incubated in DMEM+10%FCS+1%P/S supplemented with 25nM DiOC6 or 500nM TMRM, respectively, for 30min at 37°C in the dark. As a positive control for abolished membrane potential, 7.5μM of the uncoupler FCCP was added to the staining solution of three of the six tubes. Cells were analyzed on a FACS Calibur (BD Bioscience) counting 20,000 cells per sample. Dead cell exclusion was performed by gating in the FSC/SSC. Ratio of JC-1 monomers versus oligomers was detected by the ratio of mean green (FL1 channel) to mean orange/red (FL2 channel; 585 band pass filter) fluorescence. DiOC6 was detected as the mean FL1 channel intensity and TMRM as the mean FL2 channel intensity. Values were normalized to the mean of control cell triplicates and corrected for the uncoupled (FCCP-treated) control.