Membrane Permeability Assays

Membrane permeability assays were carried out using SYTOX Green Nucleic Acid Stain as in a previous study (Huang et al., 2017). Bacterial cells in log-phase were harvested and resuspended in 5% TSB in 0.85% NaCl solution to achieve the concentration of 1 × 10⁸ CFU/ml. Peptide solutions were mixed with bacterial suspension and 5 μM SYTOX green dye in the wells of a black 96 well plate. The positive control employed the permeabilized bacterial cell suspension that had been damaged by 70% isopropanol. In addition, the well-known cell lytic peptide, melittin, was also tested for the comparison. The black plate was incubated for 2 h at 37°C in the dark. The SYTOX green dye could bind with nucleoid DNA when the cell membrane was compromised. The fluorescent intensity of each well was recorded using an ELISA plate reader (Biolise BioTek EL808, Winooski, VT, United States) with excitation and emission wavelengths set to 485 and 528 nm, respectively.