Quantifying Intracellular Reactive Oxygen Species

About 3 × 10⁵ cells were harvested, washed with serum-free medium and incubated with 5 μmol/L dihydroethidium (DHE, Beyotime, Shanghai, China) at 37 °C for 30 min. Then the cells were harvested, washed and resuspended in serum-free culture medium. After staining, wash 2–3 times with PBS, replace the staining solution with fresh culture medium or buffer, and then place it under a fluorescence microscope (excitation 300 nm and emission 610 nm) (OLYMPUS, Tokyo, Japan) for observation and quantification with ImageJ software. Besides, young and senescent MSCs cultured with 10 mM N-Acetyl-cysteine (NAC, a ROS scavenger) (Selleck, Houston, TX, USA) for 24 h to test the specify of DHE [53 (link)].