Isolation and Characterization of OMVs and Exosomes

E. coli O6:K2:H1 (ATCC, Manassas, VA, USA) were cultured overnight in Luria-Bertani medium at 37  °C rotator with speed 180 rpm until the desired OD₆₀₀ was reached. The cultured E. coli were then pelleted and filtered as previously described [22 (link)], followed by OMV purification using ExoBacteria OMV Isolation Kits (SBI, Palo Alto, CA, USA), based on the manufacturer’s protocol. The protein content in the isolated OMVs was measured using Bradford assays with Coomassie Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Exosomes were isolated from the bronchoalveolar lavage fluid (BALF) of WT mice. To obtain BALF, a total of 2 mL of PBS was used for the collection, as previously described [23 (link)]. BALF was centrifuged at 300× g for 10 min to remove floating cells, followed by 2000× g for 20 min to separate apoptotic bodies, and 16,000× g for 40 min to separate microvesicles [24 (link)]. The resulting supernatants were then ultracentrifuged at 100,000× g for 1 h to obtain exosomes [24 (link)]. TEM images of OMVs were generated at the Experimental Pathology Laboratory Core, Boston University School of Medicine.