MTT toxicity assay

Human osteosarcoma U2OS cell lines were used for the cytotoxicity assay. Cells were cultured and passaged at 37 °C under 5% CO₂ in 1X medium (filtered DMEM, 10% FBS, 100 μg/ml streptomycin, and 100 μg/ml penicillin and 2 mM L-Glutamine). Cells were seeded in triplicate to clear 96-well plates with 4000 cells/well then grown for 48 h. Cells were treated with molecules at desired concentrations (final DMSO concentration 0.5%) in DMEM and incubated for 48 h. Cell viability was measured by adding tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) which is converted to insoluble purple color formazan because of the mitochondrial activity. Cells were incubated with MTT reagent for 4 h and then the medium was replaced with DMSO:EtOH (50:50) mixture. Purple salt was dissolved, and the absorbance of wells was measured at 570 nm by the spectrophotometer. As a negative control, cells treated with 5% final DMSO concentration (known as toxic to cells). In each experiment 3-technical replicates were done.