2.5. WST Assay

Cell proliferation was determined based on the metabolic activity of mitochondrial dehydrogenases using a WST-1 assay (Roche, 68305 Mannheim, Germany). In viable cells, the mitochondrial succinate tetrazolium dehydrogenase system catalyzes the conversion of the red tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzole-disulfonate) to dark red formazan, which is analyzed photometrically. In brief, cells were seeded in a concentration of 2 × 10³ cells/well of a 96-well plate, cultivated for 24 h, and stimulated with different concentration of H₂O₂ (0–200 µM) for 24 h in quadruplets. Ten microliters of WST-1 were added and cells were incubated for an additional 4 h. Optical density (OD) at 440 nm was determined relative to the OD at 650 nm using an ELISA multiplate reader (Tecan Infinite 200 Pro). The background was adjusted using 100 μL medium with 10% WST-1 reagent without cells.