JC-1 assay for evaluation of mitochondrial membrane potential

In order to examine the changes in the mitochondrial membrane polarization cell-permeant JC-1 dye was used. In the mitochondria with intact membrane, JC-1 dye easily enters and remains in the form of aggregates. Unlikely, in case of lost/decreased mitochondrial membrane potential it remains as monomers in the cytosol. Here in, MCF7 cells were incubated with 13 μM of ceranib-2 in six-well plates for 24 h. Untreated and ceranib-2 treated cells were collected and centrifuged at 1200× rpm for 5 min at room temperature. The supernatant was replaced by JC-1 working solution (0.5 mL/tube) and cells were resuspended and then incubated in a humidified CO₂ incubator at 37 °C for 15 min. MCF7 cells were washed twice with assay buffer (1×) at room temperature and pellets were vortexed in 0.5 mL of assay buffer and then analyzed by flow cytometry. A Mitochondrial Membrane Potential Detection Kit (BD Pharmingen, Franklin Lakes, NJ, USA) was used (Liu et al. 2007a, b).