Isolation and Purification of Extracellular Vesicles

Fn was cultured under anaerobic conditions (99% N2 at 37°C) until optical density (600 nm) reached 1.5 as described previously.^{18 (link)} The isolation of EVs was performed as follows. Briefly, after pelleting of bacterial cultures (10000 g for 20 min), the obtained supernatants were filtred (0.22 μm) to remove parental bacterial debris and other contaminants. The supernatant was further concentrated to 1/8 of its initial volume using 100 kDa ultrafiltration membranes (Millipore, Germany), ultracentrifuged at 4°C and 60000 g for 30 min, and washed with PBS twice to obtain the crude EVs. The purified EVs were filtered (0.22 μm) again before ultracentrifugation in a 45 Ti rotor at 150000 g at 4°C for 2 h using a sucrose density gradient, followed by endotoxin removal with a Detoxi-Gel Endotoxin Removing Column (Thermo Scientific, USA). The final pellets were resuspended in PBS and stored at −80°C.