Isolation of Outer Membrane Vesicles from E. coli

E. coli was grown in LB broth (500 mL, 37 °C, 180 rpm) until reaching an OD600 nm value of 1. Bacterial cells were removed through centrifugation at 3000× g for 20 min at 4 °C. Residual bacteria and cellular debris were eliminated by sequential 0.45 and 0.22 µm filtration (VWR Scientific, Bridgeport, NJ, USA). OMVs were collected by centrifugation at 100,000× g for 2 h at 4 °C (Optima XPN-100 Beckman Coulter and rotor 70Ti, Palo Alto, CA, USA). The vesicles were washed in sterile PBS1X through ultracentrifugation and re-suspended in 150 μL of PBS1X. LB agar plates were used to check the sterility of the OMVs. The purified vesicles were stored at −20 °C until use [28 (link)].