4.3. Cell Viability Assay

MCF7, T47D, MDA-MB-231, and MDA-MB-468 BC cell lines were placed on a 96-well plate (Nunc, Rochester, New York, NY, USA) at a density of 1 × 10⁴ cells/mL. The next day, the culture medium was removed, and cells were exposed to serial dilutions of total concentration of CAM (10–100 µM) or CDDP (0.01–10 µg/mL) individually or a combination of both compounds for 96 h. Then, the BC cell lines were incubated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution at a concentration of 5 mg/mL (Sigma) for 3 h. During this time, MTT was metabolized by living cells to purple formazan crystals, which were solubilized in a sodium dodecyl sulfate (SDS) buffer (10% SDS in 0.01 N HCl) overnight. The optical density of the product was measured at 570 nm using an Infinite M200 Pro microplate reader (Tecan, Männedorf, Switzerland). Dose–response curves were plotted to determine half-maximal inhibitory concentrations (IC₅₀) for the CAM and CDDP using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA). The results of combined treatment CAM and CDDP were analyzed according to isobolographic protocol. The drug doses were determined based on the IC₅₀ values calculated from the previous cytotoxicity test.