Mitochondrial Membrane Potential Assay

Changes in the mitochondrial membrane potential (ΔΨ_m) were estimated with the indicator rhodamine 123 (rh123). When mitochondrial membranes are polarized, the dye accumulates in the membrane and its fluorescence quenches. With calcium entry and depolarization of the mitochondria, the dye redistributes to the cytoplasm, where rh123 fluorescence intensity increases. Cultures were loaded with 10 μg/ml rh123 for 10 min and washed three times with extracellular saline plus amino acids. Z-stacks were taken with low-power 488-excitation before injury for a baseline measurement. During injury or sham, images were stream-acquisitioned at 10 Hz for 2 min to monitor the immediate effect of the injury on ΔΨ_m. After injury, stacks were taken immediately and again at 15 min post-injury. To obtain the maximal rh123 signal, mitochondria were depolarized with a 2 min treatment with the protonophoric uncoupler FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) at 0.2 μM, and a final z-stack was taken. Maximum projections of the z-stacks were computed, images were segmented into individual cellular regions, and mean intensity values were computed for each.