Ac t diff2 analyzer

Plasma hemoglobin levels were determined using a Beckman Coulter Ac∙T diff or Ac∙T diff2 Analyzer (Beckman Coulter, Inc. 11800 SW 147^th Ave, Miami, FL).

Mouse platelets were obtained following published protocols [26 (link)]. Blood was drawn from mice anesthetized with 2–3% inhaled isoflurane by cardiac puncture into 3.2% sodium citrate at a ratio of 1:9 or into heparin for the whole blood smears. To collect platelet-rich plasma (PRP), blood was centrifuged at 86 x g for 8 minutes. To increase platelet yield, the lower phase was washed 3 times with Tyrodes buffer pH7.4 (10 mM HEPES, 12 mM NaHCO3, 138 mM NaCl, 5.5 mM glucose, and 2.9 mM KCl). For washed platelets, the combined PRP was spun at 718 x 28 g for 6 minutes in the presence of prostaglandin I2 (0.1 μg/ml). The platelet pellet was resuspended in Tyrodes buffer pH7.4 (10 mM HEPES, 12 mM NaHCO3, 138 mM NaCl, 5.5 mM glucose, and 2.9 mM KCl) to the appropriate concentration. The platelet resuspension was spun at 510 x g for 30 seconds to pellet contaminating red blood cells. The platelet suspension was removed and platelet counts obtained on a Beckman Coulter AcTdiff2 Analyzer. The platelets were adjusted to concentrations optimized for the assay required and allowed to rest for 45 minutes to 1 hour before using for functional assays.