MTT Cell Viability Assay

Cell viability was measured by MTT assay (MB186, Himedia) described earlier (Ghosh-Choudhury et al., 2010; Mehta et al., 2015). In brief, cancer cells (5000 cells/well) were seeded in 96 well tissue culture plate in DMEM supplemented with 10% FBS at an atmosphere of 5% CO₂ at 37°C. After 24 h of seeding, cells were separately incubated with different concentrations [25, 100, and 250 μg/ml; equivalent to 1, 4, and 10 μl of solvents (ethanol/acetone)] of fruit pulp ethanol extract (PEE) and pulp acetone extract (PAE) for 24 h. Control cells were similarly incubated with equal volume of the solvents as in the experimental wells. At the end of the incubation period, 10 μl of MTT solution (5 mg/ml) was added to each well and the plate was incubated at 37°C for 1 h. The formazan crystals formed were solubilized by adding DMSO, and subsequently, absorbance was measured at a wavelength of 530 nm (Studzinski, 1995; Moongkarndi et al., 2004; Ghosh-Choudhury et al., 2010; Mehta et al., 2015). The effective absorbance was calculated for the experimental wells with respect to corresponding control wells. LC₅₀ (Lethal concentration at which 50% cells are killed) at 24 h duration was calculated using MTT data set for different cancer cell lines (Zhang et al., 2007).