MTT assay

MTT viability assay on HCE VK2, HFF, and HeLa cell lines using various concentrations of HPAC was performed after 24 hours of incubation. Briefly, cells were plated at a density of 1 × 10⁴ per well in a 96-well plate overnight. The following morning, concentrations starting at 10 mg/ml were twofold serially diluted and added to cell monolayers in whole media for a period of 24 hours. At the end of incubation, MTT (0.5 mg/ml in whole media) was added to cells and incubated for a period of 3 hours to allow crystal formation. Acidified isopropanol (1% glacial acetic acid v/v) was added to cells to dissolve the formazan crystals. Dissolved violet crystals were transferred to a new 96-well plate and analyzed by a microplate reader (Tecan GENious Pro) at 492 nm. MTT assay was conducted under cell-free HPAC conditions, and their values were subtracted from the cell-mediated conditions to account for signal generated through oxidative degradation of MTT by HPAC alone.