A total of 10 g of feces was inoculated into 40 mL of sterile phosphate-buffered saline (PBS) and homogenized. Next, EVs were then isolated by centrifugation as previously described, with some modifications [8 (link),13 (link),24 (link)]. A first centrifugation of the homogenate was performed (40 min, 4000× g, 4 °C), and the supernatant was recovered and filtered using sterilized vacuum filtration units, Rapid-Flow™ filters MF 75, and Nalgene® 0.2 μm in cold ice (ThermoFisher Scientific, Waltham, MA, USA). The filtrate was transferred to 10 mL polycarbonate open top thick wall tubes and ultracentrifuged at 100,000× g for 3 h at 4 °C, with a fixed angle rotor Type 70.1 Ti in a Beckman Optima XL-100K ultracentrifuge (Beckman Coulter Life Sciences, Lakeview Pkwy S Drive, Indianapolis, IN, USA). Pellets were resuspended in 200 µL of PBS and the EVs were purified using qEVoriginal size exclusion columns (SEC) of 70 nm (Izon Science Europe Ltd., Oxford, UK) [25 (link),26 (link)], following the manufacturer’s recommendations. Fractions 7–10 were collected, mixed, concentrated with Vivaspin® 20 100 kDa (Sartorius Stedim Biotech GmbH, Göttingen, Germany) centrifugal concentrators, aliquoted and frozen at −80 °C until used.
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