HepG2 cytotoxicity assay

Actively growing HepG2 cells (HB-8065, ATCC) were detached from the culture surface and dispersed with 5 ml of Eagle's Minimum Essential Media (supplemented with 10% FBS/1% NEAA solution/1% penicillin + streptomycin) by repeated pipetting. Cell suspension was added to 500 ml of the same medium at a final density of 1.2 × 10⁵ cells ml⁻¹ and 25 μl per well were seeded in 384-well-plates with pre-dispended compounds (250 nl per well) using a Multidrop combi dispenser (Thermo Scientific); this number of cells (typically 3,000 cells per well) ensures that new monolayers were not more than ∼50% confluent at the time of seeding. Cells were incubated at 37 °C and 5% CO₂ in a humidified incubator for 48 h. After incubation, plates and CellTiter-Glo Reagent (G7571, Promega) were equilibrated at room temperature for 30 min before proceeding to develop the luminescent signal. Using a Multidrop combi dispenser, 25 μl per well of the signal developer were added to the plates and after 10 min at room temperature for stabilization, plates were read on the ViewLux system (Perkin Elmer).