Van bodipy

The lectin Alexa-Fluor 647^® Wheat Germ Agglutinin (WGA-647 – Invitrogen – W32466) was suspended in sterile distilled water to obtain a stock solution at 1 mg/mL. A stock solution of Bodipy-FL Vancomycin (Van-Bodipy, Invitrogen – V34850) was prepared in DMSO at 0.5 mg/mL. Aliquotes of both stock solutions were stored at -20°C. For Gram analysis, the LIVE BacLight^TM Bacterial Gram Stain Kit (Invitrogen – L7005) was also used as recommended by the manufacturer. This kit is composed of Syto 9 and HI at 3.34 and 4.67 mM, respectively. For viability analysis, the Live/Dead^® BacLight^TM bacterial viability kit (Invitrogen – L34856) was used as recommended by the manufacturer. This kit is composed of Syto 9 (3.34 mM in DMSO) and propidium iodide (PI) (20 mM in DMSO), two nucleic acid stains that stain all bacteria and in preference bacteria with a damaged cell wall that are considered as “dead,” respectively (Berney et al., 2007 (link)).

The living cells (overnight culture) and heat-killed donor cells (log-phase) were grown/prepared as described above; Cells were washed with PBS before the staining. They were stained by the Bacterial Viability Detection Kit PI-DAPI (BS08, Dojindo, Japan) or CTC-DAPI (BS09, Dojindo, Japan) following the manufacturer's instructions (Figs. S4 and S5). BODIPY FL conjugate of vancomycin (Van-BODIPY, Invitrogen) was added to the PI-DAPI-stained cells at a final concentration of 1 μg/mL and the cells were incubated for additional 5 min at room temperature. The cells were visualized using a fluorescence microscope (BZ-X710, Keyence).