Cell Viability Assay

The CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used to determine the number of viable HepaRG™ cells based on the quantification of ATP present following each chemical treatment. Cytotoxicity was evaluated 4 h after the last exposure following the manufacturer's instructions in 96-well plates. Briefly, wells containing 100 μl cell samples were equilibrated at room temperature for 30 min prior to the addition of CellTiter-Glo^® Reagent to each well in a volume equal to that of the cell culture medium (e.g., 100 μl). The contents were mixed for 2 min on an orbital shaker to induce cell lysis prior to incubation at room temperature for 10 min to stabilize the luminescent signal. Luminescence was measured on a SpectraMax^® plate reader (Molecular Devices, San Jose, CA, USA). Luminescent signal is the result of the release of ATP from metabolically active cells and is directly proportional to the number of viable cells in the culture. The cytotoxicity cut-off was >60% cytotoxic (equivalent to <40% viable cells).