Speactramax i3x plate reader

The bactericidal activity of cationic peptides resulted from membrane disruption. DNA or RNA that leaked out of the cell was stained with SYTOX^™ Green Nucleic Acid Stain (Invitrogen Life Technologies, Carlsbad, CA, United States). The MRSA ATCC 43300 bacterial strain was used to evaluate the membrane disruptive potency of the three peptides. The assay was performed according to the procedure described previously with some modifications (Chen et al., 2018 (link)). Peptides at different concentrations (4 × MIC, 2 × MIC and 1 × MIC) were incubated with the re-suspended bacterial solution at a concentration of 5 × 10⁶ CFU/well in the 96-well black plate for 3 h without light. The positive control is 70% isopropanol. SYTOX green dye (the final concentration of 1%) was then added to each well, and the permeabilizing potency was measured as fluorescent intensity using a SpeactraMax i3x plate reader (Molecular Devices, CA, United States). All experiments were done in five replicates for statistical significance.

The membrane depolarization assay was conducted by Chakraborty’s method with some modifications (Chakraborty et al., 2012 (link)). The MRSA ATCC 43300 strain was cultured in MHB medium to the exponential phase (OD_550nm = 0.20). Then the bacterial suspension was diluted until the final bacterial density was 10⁸ CFU/ml, afterwards, the bacterial suspension was added into clean centrifuge tubes (5 ml/tube). The bacterial precipitate was harvested by centrifugation (4,500 rpm, 5 min at 4°C), then washed twice by phosphate-buffered saline. The bacteria were re-suspended by fresh MHB with different concentrations of peptides (the final concentrations of 4 × MIC, 1 × MIC and 1/4 × MIC) and incubated in a shaking incubator at 37°C for 4 h. The MRSA bacterial culture was regarded as the control group. After incubation, 90 μL of bacterial culture was transferred into black 96-wells plate together with 10 μL of rhodamine 123 fluorescent dye (the final concentration of 5 μg/ml) (Solarbio, Beijing, China). The plate was incubated in the dark for 15 min to allow the dye incorporate into bacteria. The Rh123 fluorescence was monitored by a SpeactraMax i3x plate reader (Molecular Devices, CA, United States) with the excitation and emission wavelengths of 488 and 530 nm, respectively.