2.8. Detection of Mitochondrial ROS Level Using Mito-SOX

MitoSOX (Thermo Fisher) was used to monitor mitochondrial reactive oxygen species (ROS), especially superoxide. After treatment, 5 × 10⁵ cells were mixed with 5 μM of MitoSOX reagent at 37 °C in the dark for 20 min. After harvest, the fluorescence intensity of MitoSOX was detected using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with excitation at 510 nm and emission at 580 nm [32]. The percentage change of mitochondrial ROS level by the treatment group is expressed relative to that of the control (CTR) without treatment (only medium) group.