2.5. Measurement of reactive oxygen species

Intracellular reactive oxygen species (ROS) generation was evaluated using fluorophotometric quantitation.17 Briefly, HUVECs were cultured in 24‐well plates until they grown 70% confluence. After treatments with SNP with or without DMY, the cells were incubated with 10 μmol/L DHE for 30 minutes at 37°C. Fluorescence intensity was estimated using inversion fluorescence microscope, and the relative change was processed with the ImageJ analysis program.