3.6. Cytotoxicity Assessment

For cell cytotoxicity assay the cells were passaged to 96-well plates (Falcon, clear flat bottom TC-treated, Corning) and incubated for 24 h. Afterwards, a semi confluent monolayer of appropriate cell line was treated with serial dilutions of extract stock solutions (1000–0.49 µg/mL) for 72 h. Simultaneously, the influence of DMSO, used as a solvent for stock solutions, on the cells was evaluated. Cells supplemented with 2% FBS culture media were used as a negative control. After incubation the media was removed, PBS was used to wash the cells and 10% of MTT solution (5 mg/mL) in FBS free media was added and the incubated for the next 4 h. Subsequently, the SDS/DMF/PBS (14% SDS, 36% DMF, 50% PBS) solvent was added (100 µL per well) to solubilize the formasane crystals and after overnight incubation the Synergy H1 Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA) equipped with Gen5 software (ver. 3.09.07; BioTek Instruments, Inc.) was used for absorbance measurement (540 and 620 nm). The in vitro test were done in triplicate and repeated three times. Data analysis was performed using GraphPad Prism (v8.0.1) to calculate CC₅₀ (concentration decreasing cell viability by 50% in comparison to control cells) from dose–response curves (non-linear regression). Furthermore, the selectivity indexes were calculated (VERO CC₅₀/cancer cell line CC₅₀) to evaluate the selectivity towards cancer cells. In case of VERO cells also CC₁₀ (concentration decreasing cell viability by 10% in comparison to control cells) values were evaluated for the use in further antiviral studies. Statistical evaluation was performed using GraphPad Prism (two-way ANOVA, Dunnett’s multiple comparisons test).