2.6.5. Flow cytometry detection of reactive oxygen species (ROS)

Collect cells from each group mentioned above and adjust the cell concentration to (1–10) x 10⁶ cells/mL. Add a concentration of 10 μmol/L dichlorodihydrofluorescein diacetate (DCFH‐DA), incubate at 37°C for 20 min, mix every 3–5 min to ensure thorough interaction between the probe and cells. Set cells without staining as negative controls. Wash the cells twice with phosphate‐buffered saline to remove DCFH‐DA that has not entered the cells. First, calibrate the voltage using negative control cells. Adjust the voltage at the forward scatter‐side scatter light position, encircle the desired cell cluster in the histogram, and further adjust the voltage to ensure that the negative control's x‐axis position is to the left of 10⁴. After voltage calibration, individually analyze each sample from different groups using flow cytometry.