Membrane Disruption Evaluation of Peptides

Membrane disruption evaluation was conducted by assessing ethidium bromide uptake after peptide exposure, following our previously established protocols [24 (link),39 (link),40 (link),55 (link),56 (link),57 (link),59 (link),65 (link),90 (link)]. AB5075 bacteria were cultured on Tryptic Soy agar for 18–24 h. Colonies displaying an opaque morphology were selected and suspended in DPBS (Gibco) to achieve an optical density (OD₆₀₀) of 0.1. An aliquot of 180 µL of the bacterial suspension was combined to achieve a final peptide concentration of 50 µg/mL and a final ethidium bromide concentration of 10 µM within a black 96-well flat plate (Ultracruz Poly-propylene Microplate sc-204462, Santa Cruz, CA, USA). The plate was immediately transferred to a BioTek Cytation 5 instrument for fluorescence intensity measurement every 2 min for a duration of 20 min at 37 °C (with excitation at 535 nm and emission at 590 nm wavelengths). Relative fluorescence units (RFU) were calculated as follows: RFU of tested samples minus RFU of the no-peptide control containing ethidium bromide. LL-37 and polymyxin B were employed as controls in this experimental setup. The experiment was replicated three times and performed twice. Statistical significance was determined using Student’s t-test.