4.5. Cytotoxicity Testing on Vero Cells

To measure the cytotoxic potential of the synthetic peptide toward monkey kidney epithelial cell line or Vero cells (ATCC CCL-81), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic activity assay was applied as described previously [50]. Vero cells were plated in 96-well plates at 6000 cells/well in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal bovine serum (HyClone, GE Healthcare Life Science, Logan, UT, USA) at 37 °C with 5% CO₂ using six replicates per condition. After 24 h of culture, the medium was discarded and replaced with the synthetic peptide prepared in MEM at various concentrations (0, 1, 10, and 100 µM). After 24 h of incubation, 10 µL of MTT solution (5 mg/mL in PBS, AppliChem GmbH, Darmstadt, Germany) was added and incubated for an additional 4 h at 37 °C with 5% CO₂. After medium was removed, the mixture of 4 mM HCl and 0.1% Nonidet P-40 in isopropanol was added to dissolve formazan crystals. The optical density (OD) of the dissolved formazan was measured at 590 nm, with a reference wavelength of 620 nm. The percentage of cell viability was calculated by comparing the OD of the treated groups with that of the untreated control. Descriptive statistics were used to describe the percentage viability of each treatment. Kruskal–Wallis analysis of variance (ANOVA) followed by Dunn’s multiple comparison were used to compare the differences between the treated and control groups. Significance was set at p-value < 0.05. Statistical analysis was performed using GraphPad Prism version 7.0 for Windows (GraphPad Software, San Diego, CA, USA).

To determine the effect of the peptide on programmed cell death, Vero cells were cultured with 100 µM of the synthetic peptide for 24 h, stained with fluorescein isothiocyanate (FITC)-conjugated annexin V, and counter stained with propidium iodide (Invitrogen, Eugene, OR, USA). Apoptotic and necrotic cells were visualized using confocal fluorescence microscopy (LSM 700; Carl Zeiss, Jena, Germany).