Quantification of Circulating Microparticles

Circulating endothelium-, monocyte- and platelet-derived microparticles were measured as previously reported (21 (link),22 (link)). In brief, blood samples were collected and centrifuged (160g; 20-22°C; 10 min) to obtain platelet-rich plasma (PRP). The PRP was centrifuged (1500g; 20-22°C; 6 min) to obtain platelet-poor plasma (PPP). Furthermore, PPP (50 µL) was labeled (20 min; room temperature) with CD51FITC, CD42FITC and CD31 PE, and CD14FITC (BD Biosciences) for the identification of endothelial, platelet, and monocytic microparticles, respectively. Isotypes (BD Biosciences) were used as controls. Microparticles were quantified per microliter of PPP injected into the cytometer, according to the standard protocol. TruCOUNT (BD Biosciences) tubes containing a known number of beads were used to quantify the number of microparticles per microliter of PPP.