Enhanced mitochondrial membrane potential detection kit

The enhanced mitochondrial membrane potential detection kit (C2003S, Beyotime) was employed to assess changes in mitochondrial membrane potential. Healthy mitochondria exhibited red fluorescent polymers (J-aggregates). When the mitochondrial membrane potential was reduced, it existed in the form of green fluorescent monomers (J-monomer). The staining working solution was prepared by adding 1 mL of staining buffer to 5 µL JC-1 (200X). After thorough mixing with the cells, they were incubated at 37 °C for 20 min. Following washing, 2 mL of complete medium was added, and the cells were imaged using a fluorescence microscope.

In a 6-well plate, each well was inoculated with 2 mL of cell suspension (5 × 10⁵ cells) and placed in an incubator for 24 h. The mitochondrial membrane potential of HaCaT cells was measured using the Beyotime enhanced mitochondrial membrane potential detection kit as follows: Take 50 μL of JC-1 (200X) and add 8 mL of ultrapure water. Mix well to fully dissolve, then add 2 mL of JC-1 staining buffer (5X) and mix well to prepare the staining working solution. The specific operation was conducted following the instruction manual, and the experimental results were observed and recorded using a fluorescence microscope (CKX53, Olympus LS, Tokyo, Japan).

The mitochondrial membrane potential was measured by the Enhanced Mitochondrial Membrane Potential Detection Kit (C2003S, Beyotime, China). Carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) is an uncoupler of mitochondrial oxidative phosphorylation that led to the loss of the mitochondrial membrane potential. The control group was treated with 50 μM CCCP (C6700, Solarbio, China) for 20 min, and JC‐1 fluorescence was detected.