2.4. Cytotoxicity Assay

For detailed cytotoxicity studies, Vero, HBCA, and UKF-NB-4 cells were seeded onto 96-well plates at a density of 10,000 cells per well and incubated for 24 h at 37 °C before being used in the experiment to form a confluent monolayer. After the incubation, tested compounds were added to the cells at concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 75, and 100 µM, and treated under the same regime as during antiviral testing; i.e., pretreatment for 24 h with culture medium containing appropriate drug concentrations, then the medium was exchanged with fresh compound-containing medium. After 48 h post medium exchange, cytotoxicity was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Munich, Germany) according to the manufacturer’s instructions. The 50% cytotoxic concentration (CC₅₀) values, representing the concentration of compound that reduced cell viability by 50%, were calculated using GraphPad Prism (version 7.04, GraphPad Software, San Diego, CA, USA) as a nonlinear regression (inhibitor vs. normalized response, variable slope). All assays were performed in three independent experiments done in triplicate.