Quantifying Intracellular and Mitochondrial ROS

Levels of intracellular and mitochondrial ROS were measured using a DCFH-DA or mitoSOX probe as described in the previous method [43 (link)]. In brief, at 4, 8, 16, 24, and 48 hours after corresponding treatment, cells were harvested, collected, and washed with serum-free DMEM. Then, cells were mixed with serum-free media containing 10 μM DCFH-DA probe (Molecular Probes, Eugene, OR, USA) or 5 μM mitoSOX probe (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37°C in the dark for 30 min with slight agitation every 5 min. Subsequently, cell pellets were collected, washed three times with PBS, and resuspended in 500 μl PBS for flow cytometry analysis (Cytomics FC500). The induced green fluorescence from 10,000 cells was documented at 488 or 510 nm. FlowJ software was used to analyze the average fluorescence intensity.