4.7. Laurdan Based Membrane Fluidity Assay

The peptide-induced bacterial membrane fluidity was measured by following an established protocol [41] with minor modifications. In short, the exponential phase of S. aureus MW2 bacteria was regrown from an overnight culture in fresh MHB media. Cells were then washed 3 × with PBS and resuspended in half the initial culture volume taken for washing. The Laurdan dye (Cat no. 40227, Sigma-Aldrich, Darmstadt, Germany) was added to the bacteria with a final concentration of 10 µM at room temperature in the dark. One hundred microliters of this dye/bacteria mixture was added to serially diluted peptides in a black, clear-bottom, 96-well plates (Cat no. 3904, Corning, NY, USA). After incubating in the dark for 1 h at room temperature, fluorescence intensity was measured using a spectrophotometer SpectraMax M2 (Molecular Devices, CA, USA) with excitation at 350 nm and a dual emission at 435 nm and 490 nm. The Laurdan GP was calculated using the formula GP = (I₄₃₅ − I₄₉₀)/(I₄₃₅ + I₄₉₀). 40 mM of benzyl alcohol as a membrane fluidizer, were used as a positive control.