Cytotoxicity and Metabolic Assays

To assess cytotoxicity and metabolic turnover, we used Lactate dehydrogenase (LDH) assay (i.e., cytotoxicity) or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (i.e., metabolic turnover) as previously described [28 (link),37 (link)], with minor modifications. Cells were distributed in 96-well plates (Costar, Milan, Italy) at a final density of 10,000 cells/well/100 µL and incubated for 24 h. The day after, cells were exposed to drugs as above described, and incubated for 4, 24 and 48 h. On the day of each time point, medium was removed and processed as manufacturer’s instructions for the LDH-viability assay (CytoSelectTM LDH cytotoxicity assay kit, Cell Biolabs, Milan, Italy). For metabolic turnover, MTT at a final concentration of 1 mg/mL was added to each well and incubated for 3 h under standard culture conditions. Media were then gently removed, 200 µL of MTT solvent (DMSO) was added, and cells were stirred on an orbital shaker for 10 min at room temperature. The absorbance was measured using a Varioskan Flash spectrophotometer (Thermo Scientific, Milan, Italy) at 570 nm. Data were expressed as the percentage of MTT reduction versus control cells. Each experiment was performed three times with six replicates per condition during each experimental run.