Cell Viability Assay Using CellTiter 96 AQ

The CellTiter 96 AQ_ueous assay (Promega, USA) was used to determine cell viability (8 (link)). The CellTiter 96 AQ_ueous Assay is composed of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS). RAW264.7 or HaCaT were seeded to 96-well plates (1 × 10⁴ cells/well) for 18 to 24 h. Fresh media with or without different concentrations of the test peptide were added slightly to the wells and incubated for another 24 h. The vehicle group was treated with fresh medium containing 5% water. After incubation with tested peptide, 20 μL of MTS-PMS reagent was added to each well and incubated for 1 to 4 h. The absorbance of each well was measured at 492 nm in a multimode microplate reader (Tecan, Switzerland). The experiments were repeated three times independently.