Cytotoxic Effect of Verbascum thapsus and Hypoestes procumbens

The cytotoxic effect of the V. thapsus was evaluated by colorimetric assay MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma-Aldrich), as previously reported [35 (link)]. For MTT 5 × 10³ cells/well were cultured in 96 wells and after 24 h, a fresh medium containing several concentrations of V. thapsus and H. procumbens was added. Following 24 h and 6 days of incubation, the medium was removed, LPS (1 µg/mL) (O26:B6 E. coli, Sigma-Aldhric) and Il-1β (10 ng/mL) (recombinant human Il-1ß (PeproTech EC, London, UK)) was added for 24 h. After the cells were washed and 200 µL of MTT solution (1 mg/mL in FBS-free medium) was added to each well and incubated for 2 h at 37 °C and 5% CO₂. Following 2 h of incubation, the medium was removed, each well was washed two times using cold PBS, and the formed crystals were melted using 200 μL of DMSO. The absorbance at 570 nm was read using a synergy HT plate reader (BioTek Instruments, Inc., Winooski, VT, United States).