3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for determination of cytotoxic concentration

This is a colorimetric assay that measures the reduction of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, colored (dark purple) formazan product. This assay directly assesses cell viability and requires only a small amount of test sample.[13] The cells are then solubilized with isopropanol, and the released solubilized formazan is measured spectrophotometrically.[14] Since the reduction of MTT can occur in metabolically active cells the level of activity is a measure of the viability of the cells.[15] In short, L929 fibroblastic cells were seeded in the 96-well plate and permitted to adhere for 24 h at 37°C in an incubator. Four concentrations of 100, 200, 400, and 1000 μg/ml was prepared by dissolving the extract in the medium and incubated for 48 h. After completion of 48 h, 10 μl of MTT (5 mg/ml) was added and percentage of dead cells by taking absorbance in an ELISA plate reader at 570 nm. The percentage growth inhibition (GI) was calculated using the formula given below.[16]

% GI = (100 − absorbance [sample]/absorbance [control] × 100).