Isolation of Extracellular Vesicles from Bacterial Culture

The EVs were isolated using a previously described centrifugation and filtration protocol [20 (link)], with slight modifications. Briefly, for pelleting the bacteria, the broth culture was centrifuged at 5000 × g at room temperature for 10 minutes (Centrifuge 5430 R, Eppendorf AG, Germany). For removing any remnants of intact bacterial cells, the supernatant was filtered through a 0.22 μm sterile syringe filter (Millipore, Germany). The filtrate was then re-centrifuged at 125000 × g at 4° C for 3 hours (Optima™ L-XP ultracentrifuge, Beckman, USA). The obtained pellet was suspended in 300 μl sterile phosphate-buffered saline (PBS). The EVs samples were stored at -20° C until used.