Analysis of mitochondrial membrane potential

The mitochondrial membrane potential was assessed in live primary fibroblasts using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich) as described [26]. Briefly, fibroblasts were washed and incubated with 5 µg/ml JC-1 dye for 20 min at 37℃. The cells were then rinsed with culture medium, and their images were obtained using the Applied Precision DeltaVision fluorescence microscopy system (GE Healthcare, Chicago, IL, USA). JC-1 accumulates as red fluorescent aggregates within polarized mitochondria but does as green fluorescent monomers within less polarized mitochondria.