Fitc conjugated cd14

Blood was obtained in sodium citrate and processed within 90 min at room temperature. MP counts were performed from fresh samples across the whole study. Samples were centrifuged for 10 min at 150xg and the supernatant, designated platelet-rich plasma (PRP, was then centrifuged at 13000xg for 2 min to pellet the platelets. The resulting supernatant, designated platelet-poor plasma (PPP) contained MP. A 100μl aliquot of PPP was used to assess MP by flow cytometry and the remainder was centrifuged at 17000xg for 45 min to pellet MP. The MP pellet was re-suspended in modified Tyrode’s buffer containing 0.35% BSA and stored at −70°C. MP were analyzed by flow cytometry [Becton-Dickinson LSRI (with upgraded light scatter detectors present on the machine) BD Biosciences] assessing forward and side scatter, and fluorescence using FITC-conjugated annexin V (BD Biosciences) and antibodies specific for endothelial cells ( PE-conjugated CD105 from eBioscience and PE-conjugated CD144 from BD Biosciences ), platelets (FITC-conjugated CD41 from BD Biosciences), leukocytes (PE-conjugated CD18 from BD Biosciences), neutrophils (PE-conjugated CD16b from BD Biosciences) and monocytes (FITC-conjugated CD14 from BD Biosciences). Each marker was counted separately (see supplementary data for gating strategy).