Cell Viability Assay of Resveratrol and Ascorbic Acid

The neutral red assay [23 (link),24 (link)] was used to determine the cell viability after incubation with the different test compounds. HUH7 cells were seeded at a density of 0.2 × 10⁶ cells/well (for HUH7) or 0.15 × 10⁶ cells/well (for HUH7/PON1) for 24 h and treated with 5-100 μmol/l resveratrol and 10-1000 μmol/l ascorbic acid, respectively. HUH7 cells were incubated with test compounds for 24 h, HUH7/PON1 cells for 48 h. In brief, the culture medium containing the test substances was replaced with fresh serum-containing medium including 60 μg/ml of Neutral Red (Carl Roth, Karlsruhe, Germany). After incubation for 1.5 h the medium was removed and the cells were extracted using a solution comprising 50:49:1 (v/v/v) ethanol, water and glacial acetic acid. The absorbance was measured in a plate reader (Labsystems, Helsinki, Finland) at 540 nm.