To pro 3 iodide

Propidium iodide and TO-PRO^®-3 iodide were purchased from Sigma-Aldrich^® (St. Louis, MO) and Life Technologies™ Europe B.V. (Bleiswijk, Netherlands), respectively. Fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal secondary antibody anti-human IgG1-Fc (Ab50473), rabbit polyclonal anti-HER 2 (ab2428), anti-pHER-2 (ab47263), anti-ACTIN (ab1801) antibodies and horseradish peroxidase (HRP)-conjugated goat polyclonal secondary anti-Rabbit IgG Fc (ab97200) were obtained from Abcam^® (Cambridge, UK). FITC-conjugated human IgG1 isotype control (ANC-295-040) and human IgG1 isotype control (ANC-295-010) were purchased from Adipogen^® International (Liestal, Switzerland).

Four colonies of B. cereus (ATCC 10876), E. coli (ATCC 25922), and S. aureus (ATCC 6538) were inoculated into 5 mL LB broth and incubated at 37°C for 5 h. Cells suspensions were centrifuged at 3000 × g and 25°C, washed and resuspended in phosphate buffered saline (PBS) + (0.14 M NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄ supplemented with 10 mM glucose and 0.5 mM MgCl₂; pH 7.4) to an OD₆₀₀ of 0.1. Concurrently, a blank PBS+ sample without cells and cells treated with Nisin (Sigma Aldrich) and vancomycin (Sigma Aldrich) were used as controls (36 (link)). TO-PRO-3 iodide (Sigma Aldrich) and DiOC₂(3) (Sigma Aldrich) dyes were then added into the sample and controls to a final concentration of 625 nM and 10 μM, respectively. The plates were incubated at 25°C in the dark for 5 min, and the cells were then treated with ModoCath peptides to final concentrations of 0.1×, 0.2×, and 1× MIC, respectively. Thereafter, the absorbance spectra for TO-PRO-3 iodide and DiOC₂ were determined (Supplementary Figure S1). The absorbance of the plates was read at λ_ex 640 nm and λ_em 700 nm for TO-PRO-3 iodide and λ_ex 480 nm and λ_em 530 nm for DiOC₂(3) using a fluorescence microplate reader (Gemini EM, Molecular Devices, Sunnyvale, CA, United States), where λ_ex and λ_em indicate wavelengths for excitation (λ_ex) and emission, respectively.