Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. Previous biochemical and proteomic studies have revealed that the Gram-negative bacteria-derived OMVs are composed of various components like outer membrane proteins, lipopolysaccharides, outer membrane lipids, periplasmic proteins, DNA, and RNA. Here, in this protocol, we describe the method to isolate the OMVs from the culture supernatant of Escherichia coli (E. coli).

Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. OMVs, which resemble the outer membrane and periplasm in composition, are proinflammatory and immunogenic facsimiles, and therefore could activate both innate and adaptive immunity. Here, we describe the OMVs immunization protocol and bacteria challenge protocol to induce bacterial sepsis in mice.