Cytotoxicity

The cytotoxic effect of PN peptides was assessed against HaCaT (human keratinocytes) and RAW264.7 (mouse macrophages) cells cultured in 96-well plates at a density of 2 × 10⁴ cells/well in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays were used to evaluate cytotoxicity. PN peptides (0–200 μg/ml) were added with the cells for 24 h at 37°C. Subsequently, MTT (0.5 mg/ml) was added to each well and incubated for 4 h at 37°C After incubation, formazan crystals produced was dissolved in dimethyl sulfoxide, and the absorbance at 570 nm was measured and cytotoxicity was determined as a percentage of 100% cytotoxic control (0.1% Triton X-100; Park et al., 2008). Melittin was used as the control (reference) peptide.