Cytotoxicity Evaluation of Bioactive Extracts

The cytotoxicity was tested using an MTT-based protocol [47 (link)] Briefly, the cellular monolayers in 96-well plates were incubated with serial dilutions of tested extracts in the cell media (MEM or DMEM) for 72 h. Subsequently, the media was removed, wells were washed with a sterile saline solution (PBS), media with MTT was added, and the incubation continued for the next 4 h. Subsequently, SDS/DMF/PBS mixture was used to dissolve the formazan crystals, and the next day, the absorbance was measured (540 and 620 nm) using Synergy H1 Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA) with Gen5 software (ver. 3.09.07; BioTek Instruments, Inc.). Collected data was exported to GraphPad Prism (version 7.04, GraphPad Software, Inc., La Jolla, CA, USA), and the CC₅₀ (concentration decreasing the viability by 50%) values were calculated from dose-response curves (non-linear regression model).