4.2. Peptide Cytotoxicity Assay

The cytotoxicity of the peptides against L929 murine fibroblasts was assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin were seeded in a 96-well microplate (100 μL/well, 4 × 10⁵ cells/mL) and incubated for 24 h at 37 °C in a 5% CO₂ atmosphere. Cells were then treated for 24 h with the peptides at 50, 100, 200, and 400 μg/mL in DMEM with 2% FBS. Cells in medium without peptides were used as a control. The medium was discarded and 100 µL of MTT at the concentration of 0.5 mg/mL in serum-free DMEM was added in each well. After 4 h of incubation at 37 °C, 100 μL of the proper reagent (10% Triton-X 100 in acidic isopropanol 0.1 N HCl) was added to solubilize formazan crystals formed following the reduction in MTT by viable cells, and the absorbance was measured at 540 nm. Each assay was run in triplicate. Results, from two independent experiments, were expressed as the percentage of viable cells in comparison to the control.