Mdck atcc ccl 34

MDCK (ATCC CCL-34) and Vero (ATCC CCL-81) cells initially obtained from American Type Culture Collection (Rockville, MD, USA) were seeded into 96-well plates and incubated for 24 h at 36 °C at 5% CO₂ until confluent monolayer is formed. Threefold dilutions (400–4 μg/mL) were prepared on Eagle’s minimal essential medium (MEM) from the compounds under investigation, added to the cells and incubated for 24 h at 36 °C at 5% CO₂. The cell monolayer was washed twice with saline (0.9% NaCl), and 100 μL of MTT solution [3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide], 0.5 μg/mL in MEM, was added into each well. The plates were incubated for 1 h at 36 °C, then the medium was removed and formazan pellets were dissolved in dimethyl sulfoxide (0.1 mL per well). The optical density in the wells was measured on a spectrophotometer Thermo Multiskan FC at the wavelength of 540 nm. The results obtained were used for calculating the concentration of the compound resulting in death of 50% cells in the culture (CC₅₀) using GraphPad Prism software employing the four-parameter logistic curve model. The values of CC₅₀ were then converted from μg/mL to μM.