2.5. MTT assay

The human breast cancer cell lines (MCF-7) obtained from National Centre for Cell Sciences (NCCS), Pune, India was used for MTT ((3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrzolim bromide) assay [14], [15]. The MCF-7 cell lines were grown and maintained in MEM medium in a 5% CO₂ incubator at 37 °C respectively. Further, the cell suspension was harvested by centrifugation and the adherent cells were released from their substrate by trypsinization or scraping. Later the cells were resuspended in the medium at a quantity of 1 × 10⁶ per ml. Further, a serial dilution was done to dilute the cells from 1 × 10⁶ to 1 × 10³ cells per ml respectively. Thereby, 0.1 ml of the above dilutions was plated out into the wells of a microtiter plate and control was maintained with medium alone. The cells were incubated for 12 h at 37 °C and 0.01 ml of MTT reagent (prior to the experiment MTT was dissolved in phosphate buffered saline (pH 7.4) and stored at 4 °C) was added to each well including controls and incubated for another 4 h correspondingly. Then the cells were periodically viewed under a microscope for the presence of intracellular punctuates purple precipitate. When the purple precipitate was clearly visible 0.1 ml of solubilization solution [It is a combination of 40% dimethylformamide in 2% glacial acetic acid mixed with 16% SDS (pH 4.7) and stored at room temperature] was added to all the wells, including controls and mixed well without shaking. The absorbance was recorded at 570 nm and the percentage of cell viability is calculated by using the following equation.