2.3. Measurement of mitochondrial ROS production

Cells were cultured in a 12-well plate and treated as indicated. To detect mitochondrial ROS, cells were labeled with MitoSOX™ Red superoxide indicator (Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"M36008","term_id":"214108","term_text":"M36008"}}M36008) with a final concentration of 2.5 μM for 30 min. The intensity of red fluorescence was observed and photographed under a fluorescence microscope (Carl Zeiss Axio Observer A1). Cells were then digested by 0.25% trypsin, rinsed with ice-cold phosphate buffer saline (PBS) and finally subjected to flow cytometry (BD FACSCanto II) to quantitatively determine the fluorescence intensity.