4.9. Cytotoxicity Evaluation

An MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay with the J774A.1 cell line (mouse macrophage cell line, Sigma-Aldrich) was performed to evaluate the cytotoxicity of the tested compounds. Briefly, cells (5 × 10⁴ cells/mL) in 100 µL of complete medium (DMEM high glucose supplemented with 10% fetal calf serum (FSC), 2 mM l-glutamine and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin)) were seeded into each well of 96-well plates and incubated at 37 °C and 5% CO₂. After incubation for 24 h, 100 µL of medium with various concentrations of jacoumaric acid, carasolic acid, and appropriate controls (doxorubicin and amphotericin B, purchased from Sigma-Aldrich) were added and the plates were incubated for 72 h at 37 °C and 5% CO₂. Each 96-well plate was then examined under a microscope to detect possible precipitate formation before the medium was aspirated from the well. 100 µL of MTT solution (0.5mg/mL in DMEM) was then added to each well. Cells were incubated for 2 h at 37 °C and 5% CO₂. Then, the MTT solution was removed and DMSO (100 µL/well) was added to dissolve the resulting formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min. The absorbance was measured at 570 nm with a microplate spectrophotometer (Eon Bio Tek, Winooski, VT, USA). DMSO was used as blank. CC₅₀ values were calculated by non-linear regression analysis processed on dose-response curves, using GraphPad Prism 6.0. (San Diego, CA, USA). CC₅₀ values represent the mean value calculated from three independent experiments.