MTT assay for cell proliferation

The cytotoxic efficacy of glycyrrhizinic acid was evaluated by MTT assay [13], which is a colorimetric assay based on the reduction of yellow coloured MTT by succinate dehydrogenase which is present in mitochondria. When MTT moves into the living cells, it gets reduced to insoluble formazan complex. MCF-7 cells at a density of 2 × 10⁵ cells/well were seeded in a 96-well plate, incubated for 24 h and then treated with different doses (0, 5, 10, 25, 50, 100, 200 µM) of glycyrrhizinic acid for different time periods. The untreated cells were kept as a control group. After incubation, the cells were washed with PBS twice and then 100 µl of MTT solution was added and the whole cell culture was again incubated for 50 min. Finally the absorbance was measured at 490 nm using an ELISA plate reader (ELX 800; Bio-Tek Instruments, USA).