2.8. Culture and Free Fatty Acid Induced Cytotoxicity in HepG2 Cells

Human HepG2 hepatocytes were grown at 37 °C, 5% CO₂ and 95% humidity in DMEM with high glucose and 10% FCS. Free fatty acid (FFA)-induced toxicity followed by quantitative assessment of lipid droplet formation and cytotoxicity was done in a 96-well plate and seeding of 10⁴ HepG2 in 100 µL DMEM supplemented with 10% FCS. After 24 h, medium was replaced by serum-free DMEM containing 1% BSA and 100 µM BH4, 100 or 300 µM Cp6 or vehicle (0.1% DMSO) and was incubated for further 24 h. In order to initiate FFA-induced cytotoxicity, cells were treated either with a cocktail containing DMEM supplemented with 10% FCS and a mixture of 750 µM FFAs (palmitic acid: oleic acid, 1:2) and 1% BSA or a treatment control mix containing DMEM with 10% FCS and 1% BSA. FFA media contained BH4, Cp6 or vehicle. Incubation with FFAs lasted for a maximum of 24 h, and toxicity was assessed via microscopy. After the respective incubation period, WST, SRB or Oil-Red O staining were performed (explained below).