Extracellular Vesicle Harvesting and Purification

In order to test different antibodies and experimental conditions prior to clinical use, we harvested EV from the culture of cell lines listed above. Cells were grown for 48-72 h in normal growth medium supplemented with 5% exosome-depleted FBS (Thermo, A2720801). Conditioned media was collected and centrifuged at 300 × g for 10 min to remove dead cells and debris, followed by filtration through a 0.22-μm cellulose acetate vacuum filter (Corning, 430767). Media was then concentrated to ~ 1 mL using a Centricon Plus-70 Centrifugal Filter (Millipore Sigma, UFC710008) and centrifugation at 4000 × g for 20 min, following the protocol of Lobb et al.^{33 (link)}. EV were then purified from the concentrated media using a qEV original column from IZON (iZON Science, SP1) following the manufacturer’s instructions.