The Isolation of Circulating MPs

Circulating MPs were separated according to the method reported by Dasgupta et al.²⁹ Briefly, circulating MPs were isolated by differential centrifugation. Blood was sampled by right heart puncture using a 10 ml syringe and then added to polypropylene tubes with 0.129 mol/L sodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation at 1000×g for 15 minutes at 18°C. The PRP was then centrifuged at 5000×g for 15 minutes at 18°C to obtain platelet-free plasma. The platelet-free plasma was further centrifuged using an Eppendorf 5424R centrifuge machine at 20,000×g for 30 minutes to precipitate MPs at 18°C. The pellets containing MPs were suspended in modified Tyrode buffer (MTB) and stored at −80°C.