Mitochondrial membrane potential assay kit with

Cells were seeded at a concentration of 3×10⁵ cells/mL and incubated with vehicle or notopterol at various concentrations. After 24-hr treatment, the changes in mitochondrial membrane potential (Δψm) were tested with mitochondrial membrane potential assay kit with JC-1 following the manufacturer’s instruction (Beyotimebio,Shanghai, China). Briefly, collected cells were suspended in 0.5 mL culture medium. After adding 0.5 mL JC-1 staining solution, the cells were incubated in 37°C for 20 mins. The cells were centrifugated (600× g, 4°C) for 4 mins and abandoned the supernatant. Precipitating cells were washed twice with JC-1 staining buffer (1⨰). Then, cells were resuspended with JC-1 staining buffer (1⨰) and analyzed by fluorescence microscope (Leica DM4000B, Wetzlar, Germany).

The mitochondrial membrane potential (∆Ψm) was measured using mitochondrial membrane potential assay kit with JC-1 (Beyotime) according to the manufacturer's instructions. Briefly, after indicated treatments, NRK-52E cells cultured in 6-well plates were washed by PBS and were incubated with JC-1 working solution for 20 min at 37°C. Then, cells were washed twice with JC-1 staining buffer. JC-1 stained cells were measured using a fluorescence microscope (Nikon).