Laurdan dye

We measured the peptide-induced changes to the bacterial membrane fluidity following an established protocol [15 (link)]. Briefly, we washed the exponential phase of E. faecium strain C68 bacteria three times with PBS and resuspended in half the volume of the initial culture taken for washing. To the bacterial suspensions in PBS, we added the Laurdan dye (Cat No. 40227, Sigma-Aldrich, Darmstadt, Germany) at a final concentration of 10 µM at room temperature in the dark and distributed 100 µL of this dye/bacteria mixture into 96-well transparent black plates (Cat No. 3904, Corning, New York, NY, USA) containing a 10× peptide solution. After incubation at room temperature for one hour in the dark, we measured the fluorescence intensity using a SpectraMax i3x (Molecular Devices, CA, USA), with excitation at 350 nm and dual emissions at 435 nm and 490 nm. Accordingly, we calculated the Laurdan generalized polarization (GP) using the formula GP = (I₄₃₅ − I₄₉₀)/(I₄₃₅ + I₄₉₀). Our positive control included 50 mM of benzyl alcohol as a membrane fluidizer.

The peptide-induced bacterial membrane fluidity was measured by following an established protocol [41 (link)] 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.