For mitochondrial membrane potential (ΔΨm) analysis, cells in culture medium were stained at RT with 10 µg/ml JC-1 (Beyotime Institute of Biotechnology) for 30 min before washing with binding buffer, according to the manufacturer's protocol. Cells were visualized using laser scanning confocal microscopy (FV-1000, Olympus Corporation) at x10 or x20 magnification.
The oxygen consumption rate (OCR) was assayed using an OCR assay kit (cat. no. 600800; MitoXpress ® Xtra HS; Cayman Chemical Company) according to the manufacturer's protocol. Briefly, 5x10 4 cells/well were seeded into a black, clear bottom 96-well plate (Corning, Inc.) and maintained in cell culture medium for 5 days. Subsequently, MitoXpress ® Xtra probe, 0.1 mg glucose oxidase and 10 µl antimycin A were added to each well, separately, and overlaid with HS mineral oil (MitoXpress ® Xtra HS; Cayman Chemical Company), The signal from the cells was monitored for 100 min in 10 min intervals at 37˚C, with 360 nm excitation/645 nm emission wavelengths.
The oxygen consumption rate (OCR) was assayed using an OCR assay kit (cat. no. 600800; MitoXpress ® Xtra HS; Cayman Chemical Company) according to the manufacturer's protocol. Briefly, 5x10 4 cells/well were seeded into a black, clear bottom 96-well plate (Corning, Inc.) and maintained in cell culture medium for 5 days. Subsequently, MitoXpress ® Xtra probe, 0.1 mg glucose oxidase and 10 µl antimycin A were added to each well, separately, and overlaid with HS mineral oil (MitoXpress ® Xtra HS; Cayman Chemical Company), The signal from the cells was monitored for 100 min in 10 min intervals at 37˚C, with 360 nm excitation/645 nm emission wavelengths.