Membrane Depolarization Assay with DiSC3(5)

The membrane depolarization assessment utilized the cationic dye, 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃(5)), following a modified version of our previously established method [24 (link),55 (link),56 (link),65 (link),91 (link)]. In brief, AB5075 bacterial colonies were cultured overnight on Tryptic Soy agar, and opaque colonies were dispersed in Dulbecco’s Phosphate-Buffered Saline (DPBS, Gibco) until reaching an optical density equivalent to 0.5 on the McFarland standard (~1 × 10⁸ CFU/mL). A bacterial suspension of 4 × 10⁷ CFU/mL was prepared, washed twice with DPBS, and subsequently resuspended in DPBS containing 10 µg/mL DiSC₃(5). A 100 µL aliquot of the bacteria-DiSC₃(5) suspension (n = 3) was dispensed into each well of a black 96-well flat plate (Ultracruz Poly-propylene Microplate sc-204462, Santa Cruz, CA, USA). The plate was incubated at room temperature within a BioTek Cytation 5 instrument and monitored until the fluorescence intensity stopped decreasing. Following this, a 100 µL aliquot of either peptide (final concentration of 50 µg/mL) or DPBS (untreated control) was added into each well, and the plate was promptly returned to the plate reader. Fluorescence readings were captured at 15-s intervals for a duration of 20 min (with excitation at 622 nm and emission at 670 nm). The experiment was conducted twice to ensure reproducibility of results.