4.9. Membrane Depolarization Assay

The depolarization of S. aureus ATCC 25923 cytoplasmic membrane induced by temporin-SHe and temporin-SHd was investigated using the membrane potential-sensitive cyanine dye DiSC₃(5) (3,3′-dipropylthiadicarbocyanine iodide), as detailed in [29]. An amount of 700 µL of bacteria resuspended in PBS (10⁷ cfu/mL) containing 1 μM DiSC₃(5) were preincubated in the dark during 10 min at 37 °C, and then 100 µL of 1 mM KCl were added to the mixture. Fluorescence (λex = 622 nm; λem = 670 nm) was recorded during 20 min at 37 °C (Varian Cary Eclipse fluorescence spectrophotometer) after addition of the peptide (200 µL, final concentration: 2-fold above the MIC). Three independent experiments were performed and the results correspond to a representative experiment with negative (PBS) and positive (melittin) controls.