3.2. Cell Culture and Cell Viability Assay

All chemicals, if not stated otherwise, were obtained from Sigma-Aldrich (St. Louis, MO, USA). The MCF-7, HeLa and HaCaT cell lines were purchased from Cell Lines Services (Eppelheim, Germany), the HCT-116 cell line were obtained from ATCC (ATCC-No: CCL-247). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained in a humidified atmosphere with 5% CO₂ at 37 °C in an incubator (HeraCell, Heraeus, Langenselbold, Germany). Cell viability was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay. Stock solutions of the studied compounds were obtained in 100% DMSO. Working solutions were prepared by diluting the stock solutions with DMEM medium, the final concentration of DMSO did not exceed 0.5% in the treated samples. Cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and treated for 72 h with the examined compounds in the concentration range 1–100 μM (1, 10, 25, 50 and 100 μM). Following treatment, MTT (0.5 mg/mL) was added to the medium and cells were further incubated for 2 h at 37 °C. Cells were lysed with DMSO and the absorbance of the formazan solution was measured at 550 nm with a plate reader Victor 1420 multilabel counter, (PerkinElmer, Inc. Waltham, MA USA). The optical density of the formazan solution was measured at 550 nm with a plate reader (1420 multilabel counter, Victor). The experiment was performed in triplicate. Values are expressed as the mean ± SD of at least three independent experiments