Fitc conjugated anti p selectin

Thrombin, ADP and luciferase were bought from Chrono-Log Corporation (Havertown, PA, USA). FeCl₃ was obtained from Sigma-Aldrich (St. Louis, MO, USA). FITC-conjugated anti-P-selectin and FITC-conjugated annexin V were purchased from BD Biosciences (Franklin Lakes, NJ, USA), while PE-conjugated Jon/A was purchased from Emfret Analytics (Würzburg, Germany). Research cigarettes (3R4F) were purchased from the University of Kentucky.

PRP was prepared in sodium citrate and stimulated with thrombin (in the presence of 10 µM Gly-Pro-Arg-Pro-NH₂), CRP, ADP, or U46619 for 20 min at 37 °C in the presence of FITC-conjugated anti-P-selectin (BD Biosciences), PE-conjugated JON/A (Emfret, Würzburg, Germany) and APC/Cy7-conjugated anti-CD63 (Biolegend) antibodies. Platelets were subsequently fixed and analyzed by fluorescence-activated cell sorting (FACS) using an LSRFortessa cell analyzer (BD Biosciences) and FlowJo software.
For receptor expression studies, PRP was incubated with FITC-conjugated antibodies directed against surface membrane glycoproteins GP1b (CD42b), GPVI, integrin α2 (CD49b) (Emfret, Eibelstadt, Germany), integrin αIIb (CD41) (BD Biosciences, Oxford, UK), or PE-conjugated antibodies against integrin β2 (CD18) (Biolegend). Receptor expression was also studied upon stimulation with 0.1 U/mL thrombin for 20 min at 37 °C in the presence of 10 µM Gly-Pro-Arg-Pro-NH₂. Platelets were subsequently analyzed by FACS.