2.7.2. Cytotoxicity assessment (MTT assay)

The vitality of the cultured cells was investigated using enzymatic reduction of the tetrazolium dye MTT to formazan. To evaluate anticancer activity, three cell lines were used, including two tumor cell lines (MCF-7: a human breast cancer cell line, and HELA: cancerous cervical tumor cells) and a normal cell line (HEK293: human embryonic kidney cells). The three cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.2 % sodium bicarbonate and 10 % fetal bovine serum (FBS). The cells were maintained in a 5 % CO₂–95 % atmosphere under high humidity at 37 °C. Cells were assessed for cell viability by the trypan blue dye exclusion assay as described earlier [24]. Batches of cells showing more than 98 % cell viability were used in this study, where cell viability was assessed using the MTT assay as described previously [25]. Briefly, 10,000 cells were plated in 96-well culture plates and allowed to adhere for 24 h in a CO₂ incubator at 37 °C. After the exposure, MTT (5 mg/ml of stock PBS) was added (10 μl/well of 100 μl of cell suspension) to each well, and the plates were incubated further for 4 h in a CO₂ incubator. Then, the supernatant was discarded, and 200 μl of DMSO was added to each well and mixed gently. The developed color was read at 550 nm in an Evolution 201 reader (Thermo Scientific, Waltham, MA, USA). Untreated control sets were also run under identical conditions. All the values were corrected from background absorbance.

The survivability percentage x100% was calculated using (ODt/ODc), where ODc denotes the density of untreated cells and ODt signifies the density of wells treated with the test compound. By graphing the association between the number of cells that survived and the quantities of compounds, survival curves for each cancer cell line and the normal cell line following treatment with the specified substance were constructed. Using concentration chart pad Prism software, the 50 % inhibitory concentration (IC₅₀) was determined from charts of the dose response curve for all (San Diego, CA, USA).