4.9. Membrane Permeability Kinetic Assay

The membrane permeability activity was evaluated by the SYTOX™ Green nucleic acid stain (Life Technologies, Carlsbad, CA, USA). S. aureus was inoculated in TSB overnight, and the subculture was conducted in the same medium for 2.5 h to reach a logarithmic growth phase. Then, the culture was centrifuged to collect the cells at the bottom of the tube, and 30 mL of 5% TSB in 0.85% NaCl were used to wash the bacterial cells twice. The concentrations of 8, 16, 32, and 64 µM were selected as the working concentrations. Then 40 µL peptide solutions were mixed with 50 µL bacterial cultures and 10 µL of SYTOX Green. Then 8 µM melittin and 5% TSB were utilised as the positive and negative controls, respectively. The fluorescent intensity of each well was tested at 37 ℃ with the excitation at 485 nm and emission at 528 nm by a microplate reader (EL808, Biolise BioTek Winooski, Minneapolis, USA) for 2 h and the interval was 5 min.