Quantifying Cellular ROS Generation

The dihydroethidium (DHE) probe (Beyotime, Shanghai, China) was used to measure ROS generation in H9c2 cells, as previously described [13 (link)]. Briefly, cells were adjusted to 1.5×10⁵ cells per well and cultured in a 24-well culture plate. After hypoxic or normoxic treatment, the culture media was removed, and cells were washed three times in PBS. The DHE probe (10 μmol/L) was added to the wells for 30 min at 37°C. Photomicrographs were taken and the mean fluorescence intensity (MFI) was calculated using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and ImageJ software (National Institutes of Health, Bethesda, MD, USA), respectively.