Cytotoxicity test against RAW macrophage cell line using MTT assay

A murine macrophage cell line (RAW 264.7) was cultured in DMEM medium supplemented with penicillin G sodium (100 μg/mL), streptomycin sulfate (100 μg/mL) and 10% FBS. Macrophages at a density of 1 x 10⁶ cells/mL were incubated at different concentrations (ranging from 0.39–100 μg/mL) of the compounds for 48 hours in a CO₂ incubator (5% CO₂, 37° C). Macrophages without any treatment were taken as negative control and amphotericin B was used as positive control. At the termination of the assay, MTT was added and incubated for an additional 3 hours. Acidified isopropanol was added to the set-up to solubilize the formazan crystals formed by the viable cells. The intensity of formazan formation was read on an Elisa plate reader (Varioskan Lux Elisa microplate reader, Thermo Fischer Scientific, USA) at 570 nm. The 50% cytotoxic concentrations (CC₅₀) of the compounds were determined by analysis of dose-response curves. Selectivity index or therapeutic index was calculated as a ratio of CC₅₀ RAW 264.7/IC₅₀ L. donovani promastigotes or amastigotes [11, 12].