4.4. Quantification of Intracellular Reactive Oxygen Species

The generation of intracellular reactive oxygen species (ROS) in the endothelial cells was performed as previously described [40]. After 24 h of incubation, the medium was removed and replaced with 10 μM H₂DCFDA (Sigma Aldrich), and the cells were incubated for 45 min at 37 °C. The fluorescence was measured after 90 min using a FilterMax F5 microplate reader (Thermo Fisher Scientific) at a maximum excitation of 485 nm and emission spectra of 530 nm. The concentrations of flavonoids were 150, 200, and 250 μM.