Measurement of mitochondrial membrane potential (JC-1)

The cells were inoculated into the well plates at a density of 1×10⁴ cells/well, placed in an incubator for 24 h, and treated with drug for another 24 h. The cells were rinsed twice with saline, 500 µl JC-1 working solution was added to each well, and the plates were placed in an incubator (5% CO₂, 37°C) and incubated for an additional 20 min. The staining solution was discarded, and the cells were rinsed with saline twice, then stained with DAPI solution at room temperature in dark for 15 min. As JC-1 aggregates in the mitochondria when the mitochondrial membrane potential is high, red fluorescence is emitted; conversely, when mitochondria are depolarized, JC-1 is in a monomer state and green fluorescence is emitted. Thus, the mitochondrial membrane potential of the cell can be judged according to the ratio of red and green fluorescence, and change of JC-1 fluorescence color can indicate change of the mitochondrial membrane potential. A decrease in mitochondrial membrane potential is considered a sign of early apoptosis (29). Red and green fluorescence were analyzed with a flow cytometer (BD FACSCalibur; BD Biosciences). The results were analyzed with CellQuest Pro 5.1.1 (Becton, Dickinson and Company).