Assessment of mitochondrial membrane potential by JC-1 dye

JC-1 is an ideal fluorescent probe that is widely used to detect MMP, △Ψm. JC-1 dye accumulates in the mitochondria in a potential-dependent manner and can be used to detect cells, tissues or purified mitochondrial membrane potential. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form polymers, which emit intense red fluorescence (Ex = 585 nm, Em = 590 nm), while the JC-1 that is present in the cytoplasm will exist in monomer form and emit green fluorescence (Ex = 514 nm, Em = 529 nm) due to the down-regulation or loss of membrane potential. JC-1 be used for qualitative detection due to the color change that directly reflects the change of MMP, and it can also be used for quantitative detection due to the degree of mitochondrial depolarization, which can be measured by the ratio of red/green fluorescence intensity. The cells were planted in 12-well plates at a density of 5 × 10⁵ cells/ml and grown in an incubator containing 5% CO₂ at 37 °C overnight. Appropriate compounds were chosen for apoptosis induction according to the specific desired procedures. A 0.5 ml cell suspension sample was transferred to a sterile centrifuge tube and centrifuged at 400 × g for 5 min at room temperature, and the supernatant was discarded. The cells were resuspended with 0.5 ml of JC-1 working solution (Yeasen, Shanghai, China) and incubated for 15–30 min at 37 °C with 5% CO₂, followed by centrifugation at 400 × g for 5 min at room temperature. The supernatant was then discarded. The cells were then washed two times by resuspending 2 ml of cell culture media or buffer and centrifuged at 400 × g for 5 min at room temperature, and the supernatants were discarded. Finally, the cells were resuspended with 0.5 ml of fresh medium or buffer for subsequent flow analysis. Healthy mitochondria containing red JC-1 aggregates were detected with the PE channel, and apoptotic or unhealthy cells containing green JC-1 monomer were detected with the FITC channel.