Membrane Depolarization Assay

The cytoplasmic membrane depolarizing activity of the peptides was evaluated using the potentiometric dye DiSC₃(5).^55,56 Stationary phase bacterial cell suspensions were washed, resuspended in 5 mM HEPES (20 mM glucose, pH 7.2) buffer to ∼10⁶ CFU/mL, and preincubated with 2 μM DiSC₃(5) for 30 min. Upon uptake into live cells, the fluorescence of DiSC₃(5) gets quenched, and in the presence of a membrane depolarizing agent, the fluorescence detected would be higher, reflecting the release of the dye into the surrounding environment. The dissipation of membrane potential of the dye-loaded cells upon addition of increasing peptide concentrations (5 to 40 μM) was observed using a Shimadzu RF-5301 PC spectrofluorometer (Ex: 622 nm and Em: 669 nm, 3 nm slit width). The corresponding killing was also determined through a colony count assay after exposing the dye-loaded cells to 20 μM concentration of each peptide and plating aliquots onto agar plates within ∼2 min.