4.6. ROS Assay

ROS generation was measured using the DCFH-DA dye. The cells were seeded at a density of 60,000 per well of 96-well plate and grown for 12 h. After that, the medium was replaced with a fresh one with 25 μM of the dye, and the cells were incubated in the CO₂ incubator at 37 °C for 1 h. After the incubation, the cells were washed twice with the culture medium and treated with the substances in the culture medium for 1 h at 25 °C. Cells treated with medium without H₂O₂ and substances were used as a control. After the incubation, the cells were washed twice with Hanks’ balanced salt solution with 25 mM HEPES and 1 mg/mL fatty acid-free bovine serum albumin, pH 7.4, and the fluorescence was measured using the plate reader Hidex Sense Beta Plus (Hidex, Turku, Finland), λ_ex = 490 nm, λ_em = 535 nm.