4.4. MTT Viability Assay

The cytotoxic activity of doxorubicin and the combined effect of doxorubicin and cucumarioside A₂-2 were evaluated using the MTT method. For this purpose, 5 × 10⁴ cells/well were seeded in 96-well microplates, then 20 μL of DOX solution at different concentrations was added and microplates were incubated for 48 h in a CO₂-incubator at 37 °C and 5% of CO_2. To study the combined effect, 20 μL of cucumarioside A₂-2 solution at different concentrations was added to wells first, and EAC cells were incubated during 2 h for blocking of MDR. Then 20 μL of DOX (25 μM final concentration) was added, and cells were incubated for 48 h at 37 °C and 5% of CO₂. Then the medium was replaced with 100 μL of fresh medium containing 10 μL of MTT solution (Sigma, USA) and microplates were incubated for another 4 h. After that 100 μL SDS-HCl solution was added and microplates were incubated again at 37 °C for 4–18 h. The optical density was measured with a microplate spectrophotometer Multiskan FC (Thermo Scientific, Canada) at 570 nm. The cytotoxic activity of the substances was expressed as percent of cell viability in comparison with the control.