Mica discs

RJEV shapes were confirmed using AFM, as previously described.⁵⁸ (link) Briefly, freshly cleaved mica discs (12-mm diameter; Electron Microscopy Sciences, US) were coated with 50 μL of a 0.1 M solution of poly-L-lysine (PLL) for 5 min. Subsequently, discs were washed three times with double-distilled H₂O (ddH₂O) and dried with a gentle stream of N₂. RJEVs were then immobilized onto the PLL-coated mica by incubation for 30 min at room temperature. Samples were covered to avoid drying and potential dust contamination. After incubation, specimens were washed three times with ddH₂O and mounted onto an Asylum MFP 3D-SA AFM (Asylum Research, US). Samples were analyzed in intermittent contact mode (alternating current [AC] mode) with TAP300GD-G cantilevers (BudgetSensors, Bulgaria), obtaining height, amplitude, and phase channel images of substrates under environmental conditions. From the resulting height images, the surface profiles, maximum and mean vesicle height, and RMS roughness of RJEVs were calculated in the Gwyddion 2.56 software. RJEVs from three independent sample preparations were utilized for all AFM-based experiments.

For AFM-based experiments, freshly cleaved 12-mm diameter mica discs (Electron Microscopy Sciences, US) were coated with 50 µL of a 0.1 M solution of poly-L-lysine (PLL, Sigma) for 5 minutes, washed 3x with ultrapure H₂O and dried under a gentle stream of N₂. Subsequently, a 50μL droplet of either HEc-EVs solution or bacterial suspension was incubated on the PLL-coated mica for 30 minutes at room temperature. Substrates were then washed 3x with ultrapure H₂O, and experiments were carried out on the AFM immediately after sample preparation.