2.11. MTT Assay

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was purchased from Sigma-Aldrich (Saint Louis, MO, USA). HepG2 cells were seeded onto 96-well plates (5000 cells/well), using DMEM. After 24 h, the cells were incubated with palmitic acid (PA; 400 μM), chenodeoxycholic acid (CDCA; 100 μM), a combination of CDCA and PA, and DMSO (mock control) for 24 h. CDCA is one of the most toxic bile acids with hydrophobic profile that can induce hepatotoxicity [13,14,15]. After treatment incubation, MTT solution (0.25 mg/mL MTT in PBS, pH 7.4) was added to each well, followed by incubation at 37 °C for 4 h. The supernatants were removed from wells, and DMSO was added to dissolve crystals. The optical density was measured at 540 nm, using an ELISA microplate reader (Tecan, Research Triangle Park, NC, USA).