3.4. Assay Protocol for Cytotoxic Activity

Cytotoxic activity was performed according to the previously described methods [56,57]. Breast cancer cell lines MDA-MB-231 were maintained in Dulbecco’s modified eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA). The media was supplemented with 10% fetal bovine serum (FBS) and 1% antimycotic antibiotic (Invitrogen, Carlsbad, CA, USA). Cells were cultured in a 5% CO₂-humidified atmosphere at 37 °C. Stock solution (5 mg/mL) of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Merck, Darmstadt, Hesse, Germany) was prepared in phosphate-buffered saline (PBS).

Cells were seeded at a density of 1 × 10⁴ cells per well in 96-well microtiter culture plates. After overnight incubation, normal growth medium was removed and replaced with either fresh medium (untreated control) or different concentrations of respective compounds in growth medium diluted from a 2 mM stock. After 24 h of incubation, MTT solution was added to each well (0.1 mg/mL in DMEM) and incubated further for 4 h at 37 °C. Upon termination, the supernatant was aspirated and the MTT formazan, formed by metabolically viable cells, was dissolved in a solubilization solution containing DMSO (100 սL) by mixing for 5 min on a gyratory shaker. The absorbance was measured at 540 nm (reference wavelength 690 nm) on an Ultra Multifunctional Microplate Reader (Bio-Rad, Hercules, California, USA). Absorbance of control (without treatment) was considered as 100% cell survival. Each treatment had three replicate wells [56]. The IC₅₀ values of the active compounds were calculated using nonlinear regression through GraphPad Prism 4 software (La Jolla, CA, USA).