4.5. Antiproliferative Assay

The effects of the synthesized compounds on cell viability were determined using the MTT colorimetric test. All examined cells were diluted with the growth medium to 3.5 × 104 cells per mL, and the aliquots (7 × 103 cells per 200 μL) were placed in individual wells in 96-multiplates (Eppendorf, Germany) and incubated for 24 h. The next day the cells were then treated with synthesized compounds separately at the final concentration of 75 μM and incubated for 72 h at 37 °C in a 5% CO₂ atmosphere. After incubation, the cells were then treated with 40 μL MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg mL⁻¹ in PBS) and incubated for 4 h. After an additional 4 h incubation, the medium with MTT was removed, and DMSO (150 μL) was added to dissolve the crystals formazan. The plates were shaken for 10 min. The optical density of each well was determined at 560 nm using a microplate reader GloMax Multi+ (Promega, Madison, WI, USA). Each of the tested compounds was evaluated for cytotoxicity in three separate experiments.