Cytotoxicity assays of test compounds against mammalian cells

Two assays were used to assess cytotoxicity: MTT assay and enumeration of host cell nuclei. Reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to measure mammalian (THP-1) cell viability in the presence of all assayed compounds at the end of a three-day incubation period with each of the compounds. MTT was added to cells at a final concentration of 0.5 mg/ml and the cells incubated for 4 hours at 37 °C in a humidified incubator at 5% CO₂. The medium was replaced with 100 μL isopropyl alcohol containing 40 mM HCl and 20 μL of 10% sodium dodecyl sulfate to solubilize precipitated serum protein. Absorbance of the reaction product was measured at 570 nm in a Tecan Infinite 200 PRO microplate reader. Compounds that decreased reduction of tetrazolium dye by ≥20% of control in an average of two independent experiments were deemed cytotoxic. As a second cytotoxicity assay, we used the established method of nuclei counts per field of view. Briefly, we measured the number of Hoechst-stained nuclei per image in Cell Profiler (v2.1.1) and expressed the nuclei number as an average count from ≈7 images per treatment ± standard deviation from a single experiment.