Mitotracker red cmxros staining

The MitoTracker Red CMXRos Staining and LysoTracker Green Staining kits were purchased from Beyotime Biotechnology, Shanghai, China. According to the manufacturer’s recommendation, the cells were incubated with MitoTracker Red CMXRos dyeing solution for 30 min at 37 °C. The cells were washed with PBS and subjected to microscopy.

The cells were incubated with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime), and DMSO was added as the control. Then, the cells were analyzed by flow cytometry.
Mitochondrial activity was analyzed using MitoTracker Red (CMXRos) staining (Beyotime). The cells were stained with the dye and then analyzed by flow cytometry. The mean fluorescence intensity (MFI) of tiplaxtinin-treated cells was compared with untreated control cells.
Mitochondrial membrane potential was analyzed with a mitochondrial membrane potential detection kit (Beyotime). The cells were stained with JC-1 and were analyzed by flow cytometry. The fluorescent emission of JC-1 aggregates (590 nm, red) and that of JC-1 monomers (520 nm, green) were measured, and the ratio of the fluorescent intensities of aggregate to monomer dyes was determined.

Hoechst33342 and MitoTracker Red CMXRos staining were purchased from Beyotime (Shanghai, China). After intervention, AR42J cells were incubated in a 2 mL High-Glucose DMEM with 20 μL Hoechst33342(10 μmol/L) and 2 μL MitoTracker Red CMXRos staining (200 nmol/L) in each plate for 15 min at 37°C as ordered by the manufacturer's instructions. 2 mL of complete medium was supplemented after being washed twice with D-PBS. The cells were visualized under a Leica confocal laser scanning microscope (EL6000, Wetzlar, Germany). Hoechst33342 was monitored at an excitation wavelength of 346 nm, and MitoTracker Red was monitored at an excitation wavelength of 579 nm to locate nuclear and mitochondria, respectively.