4.5. Bacterial Cell Membrane Depolarization Assay

Membrane depolarization was performed using the membrane potential-sensitive DiSC3(5) fluorescent probe as described previously [41,55]. After interaction with intact cytoplasmic membrane, the fluorescent probe DiSC3(5) was quenched. After incubation with the antimicrobial protein or peptide, the membrane depolarization was induced the probe was released to the medium, ensuing in an increase of fluorescence that can be quantified and monitored as a function of time. Bacterial cultures were grown at 37 °C to an OD₆₀₀ of 0.2, centrifuged at 5.000× g for 7 min, washed with 5 mM HEPES-KOH, 20 mM glucose (pH 7.2), and resuspended in 5 mM HEPES-KOH 20 mM glucose 100 mM KCl (pH 7.2) to an OD₆₀₀ of 0.05. DiSC3(5) was added to a final concentration of 0.4 µM, and changes in the fluorescence were continuously recorded after the addition of protein (from 0.01 to 20 µM) in a Victor3 plate reader. Effective dose values (ED₅₀) were estimated from nonlinear regression analysis.