Vetscan hm2 hematology analyzer

This study was approved by the institutional review board at Osaka University Hospital. For in vitro experiments, platelets were isolated from the venous blood of one healthy volunteer. The platelet preparation method was adapted from Ford et al. [14] . Five vol of OptiPrep TM was diluted with 22 vol of Solution B [0.85 % (w/v) NaCl, 20 mM Hepes-NaOH, pH 7.4, 1 mM EDTA] to produce a 1.063 g/ml density barrier. In a 15-ml centrifuge tube, 5 ml of blood was layered over 5 ml of the 1.063 g/ml solution and centrifuged at 350 g for 15 min at 20 °C in a swingingbucket rotor, and the rotor was allowed to decelerate without braking. The platelet-containing band was harvested from the broad turbid band below the interface. Isolated platelets were counted in a Vetscan HM2 Hematology Analyzer (Abaxis) and suspended in RPMI 1640 for concentration adjustment. Cytokines in the culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). Platelet-derived growth factor (R&D Systems, Minneapolis, MN, USA), transforming growth factor-b (R&D Systems, Minneapolis, MN, USA), and prostaglandin E2 (ENZO Life Science, Famingdale, NY, USA) were measured using an ELISA kit, and interleukin-6 was measured by LSI Medience Corp. (Tokyo, Japan).