Platelet Isolation and Extruded Nanovesicle Production

Platelets were isolated from the blood of volunteers according to reported protocols (11 (link)). The whole sample of fresh blood was centrifuged at room temperature at 100 × g for 20 min. After centrifugation, the whole blood sample was divided into three layers, from top to bottom: the plasma layer, white blood cell layer, and red blood cell layer. The pale-yellow plasma layer was collected and centrifuged at 800 × g for 20 min. After centrifugation, the pellets were platelets. The pellet was resuspended in 1 μM PGE1 in phosphate buffered saline to inhibit platelet aggregation and stored at room temperature until use. The platelet solution of 1 × 10⁶ cells/mL was squeezed six times with 400nm, 200nm, and 100nm pore size polycarbonate membrane filters (Whatman) in a micro extruder (Avanti Polar Lipids), and NPVs were obtained after centrifugation.