Isolation of Extracellular Vesicles via Ultrafiltration

EVs were isolated from the media of cells following a 72 h incubation using a modified ultrafiltration procedure as previously described^{46 (link)}. Briefly, 100 mL of spent media per collection was sequentially passed through 0.22 µm PES and 0.1 µm PVDF syringe filters (Merck Millipore, Kilsyth, VIC, Australia). Clarified media was then placed in a 100 kDa centrifugal filter unit (Merck Millipore) and centrifuged at 4,000 × g, 4 °C until sample had passed through. The flowthrough was discarded and the retentate washed with 0.1 µm filtered sterile PBS twice to yield a retentate of concentrated EVs in PBS. Retentates were collected and stored at −30 °C until required.