Zen 2.1 blue edition

Biofilms were grown in confocal dishes (SPL Life Sciences, South Korea) at 37°C for 24 hr in LB broth. The biofilm cells were stained with FilmTracer SYPRO Ruby (Invitrogen, USA) for 30 min at room temperature (RT), protected from light, and then washed with distilled water. The obtained CLSM (Carl Zeiss, Germany) images were analyzed and modified using the Zen 2.1 (Blue edition; Carl Zeiss, Germany) software. LB broth in sterile 96-well microtiter plates (SPL Life Sciences) was inoculated in triplicate with each overnight LB-grown culture and further diluted 1:100 with LB broth. The volume of the cells was determined by converting the OD₆₀₀ value of the O/N cells. Uninoculated LB broth was used as the negative control. The microtiter plates were then incubated at 37°C for 24 hr. After removing planktonic cells, the biofilm biomass was stained with crystal violet and solubilized with 95% ethanol (v/v), after which its absorbance was measured at 595 nm. To measure OMVs-induced initial stage of biofilm formation in Lab-WT, cells were grown at 37°C in LB until an OD₆₀₀ reached around 0.6 (for 6 hr). Biofilm formation was checked after additional 4 hr incubation with PMR^High-driven OMVs (0, 12.5, or 25 μg/mL), then both crystal violet assay and CLSM observation were used.