HEK-293 Cytotoxicity Assays

HEK-293 cell lines were seeded in 100 µL aliquots into 6.25 mm diameter culture wells at a density of 180,000 cells mL⁻¹. Cells were seeded in RPMI medium supplemented with 1× glutaMAX, 10% fetal calf serum and 1% penicillin/streptomycin. Cells were left to adhere in a humified incubator at 37°C, 5% CO₂ for approximately 16 h before being challenged with 100 µL of RPMI medium supplemented with 1× glutaMAX ± 2× the final metronidazole concentration. Cells were incubated at 37°C, 5% CO₂ for 48 h. Following challenge, 10 µL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent (Promega) was added to each well (and also to a medium-only containing well to allow for baseline medium absorbance subtraction) and cells were incubated for 1 h further at 37°C, 5% CO₂. Absorbance of wells was measured at 490 nm, and the absorbance value of the media-only control well was subtracted from all other measurements. The baseline-subtracted absorbance of challenged wells was compared to that of an unchallenged control well to determine percentage cell viability for each metronidazole concentration. IC₅₀ values (the concentration of compound at which viability was reduced by 50% relative to the unchallenged control) were calculated using a dose-response inhibition four-parameter variable slope equation in GraphPad Prism 7.0 (GraphPad Software Inc). Calculated IC₅₀ values are the averages of at least three biological replicates.