Isolation and Quantification of Mouse Plasma Microvesicles

Mouse venous blood was collected by retro-orbital puncture under anaesthesia in 0.129 mol/L sodium citrate (ratio of blood to anticoagulant 4:1). Samples were centrifuged at 1500xg for 15 min at room temperature. Harvested, platelet-poor plasma samples were further centrifuged at 18,000xg for 5 min, twice, to achieve platelet-free plasma. Platelet-free plasma was centrifuged at 18,000xg for 45 min, the supernatant removed and retained, and the pellet centrifuged for a further 45 min at 18,000xg in a solution of sodium citrate and PBS (ratio of citrate to PBS 1:3) to pellet MV. Total MV numbers were quantified by detection of phosphatidylserine using FITC-Annexin V labelling (Beckman Coulter, Brea, CA), as previously described^{41 (link)}. Samples were acquired on a Gallios flow cytometer (Beckman Coulter) and analysed using Kaluza (Beckman Coulter).