Xn 5 hematology analyzer

Healthy volunteers underwent phlebotomy with 19 gauge needles without the use of tourniquets into sodium citrate-containing tubes (BD Vaccutainer, Franklin Lakes, NJ). Washed platelets were isolated as previously described [10 (link), 15 (link)]. In short, blood collected in tubes containing sodium citrate was centrifuged (200g × 10min) to obtain platelet-rich plasma (PRP). PRP was centrifuged (1000g × 10min) and the platelet pellet resuspended in Tyrode’s buffer (139mM NaCl, 3mM KCl, 17mM NaHCO₃, 12mM glucose, 3mM CaCl₂, 1mM MgCl₂). Platelets were then counted on a XN-V Hematology Analyzer (Sysmex, Kobe, Japan), centrifuged again at 1000g × 10min, and the Tyrode’s buffer supernatant was removed. Washed platelets were then resuspended in RPMI 1640 at a concentration of 1×10⁶ platelets/μL. Platelets were kept at 37°C for 60 minutes and then centrifuged at 2500g × 5 min. The supernatant was removed and passed through a 220nm filter. The filtered supernatant (platelet conditioned media – PCM) was then employed in experiments as described below. Approximately 50–60% of platelets used in making PCM exhibited surface P-selectin at the completion of the protocol.