Cytotoxicity Evaluation of Compound A11 on L929 Cells

The cytotoxicity of A11 was evaluated by the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay^{17 (link)}. L929 mouse fibroblast cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% (v/v) of 100 IU/ml penicillin/streptomycin and 10% (v/v) fetal bovine serum (FBS). The cells were maintained in an air atmosphere containing the 5% CO₂ under high humidity at 37 °C. L929 cell lines were seeded into a sterile 96-well plate (1 × 10⁴ cells/well) then incubated overnight and treated with different concentrations of A11 (0.98–250 µg/ml) for 24 h. Media were removed, and cultured cells were incubated with 100 µl of MTT solution (4 mg/ml) for 4 h at 37 °C. After removal of MTT solution, the formed formazan was dissolved in 100 µl DMSO. Absorbance was measured by microplate reader at 570 nm; untreated cells served as negative control and melittin-treated cells as a positive control. Cell viability was determined following this equation: $$\text{Cell viability}\left(\%\right)=\left({\text{OD}}_{\text{Treated}}/{\text{OD}}_{\text{Control}}\right)\times 100$$