Sodium citrate dehydrate

Bulk phase activation of the platelets was measured by flow cytometry using previously described methods [25 (link)]. Briefly, blood was sampled from the exit port of the parallel plate chamber at two points in time: immediately after the sample initially crossed the test surface (determined visually) and also after 5 min perfusion. Aliquots of these samples were incubated in buffer solution (Tyrode’s solution with 1% BSA, Electron Microscopy Science, Hatfield, PA, USA) with fluorescein isothiocyanate – conjugated mouse anti–human CD42b (CD42b, clone LG.3A10; AbDSerotec, Raleigh, NC, USA) and either recombinant phycoerythrin–conjugated (RPE) mouse anti-human CD62P or RPE-conjugated isotype- matched control antibody (CD62P and Mouse IgG₁, clones Psel.KO.2.5 and W3/25, respectively; both from AbDSerotec) in the dark for 20 min. The cells were washed with Tyrode’s solution with 1% BSA and 0.106 M sodium citrate dehydrate (Thermo Fisher), centrifuged, and the pellet isolated. The cells were re-suspended in 1% paraformaldehyde (Sigma-Aldrich) and stored at 4° C (<24 hr) until analyzed. CD42b+ events (>5000) were collected on a 3-color FACScan (BD Biosciences) and analyzed with WINList software (Verity Software House, Topsham, ME).