Measurement of ROS generation

According to the manufacturer's protocol, the ROS assay kit (S0033; Beyotime Institute of Biotechnology, Inc.) was used to measure intracellular ROS levels using the probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA). Briefly, cells treated with 100 µM H₂O₂ for 2 h or 100 µM H₂O₂ for 2 h + 200 µM vitamin C for 48 h were washed twice with PBS. The cells were then incubated with 10 µM DCFH-DA at 37°C for 30 min. After washing with PBS twice, the fluorescence of the cells was imaged using a confocal microscope (Carl Zeiss AG) with an excitation of 488 nm/emission of 529 nm. The fluorescence intensity was measured using ImageJ_v1.8.0 software.