Cd9 pe

For flow cytometry activation experiments, washed platelets were stimulated with thrombin (#13188, Cayman Chemicals), 2-meADP (#1624, Tocris), and convulxin (#sc-202554, Santa Cruz) for 10 min. and then CD62P antibody (#553774, BD Biosciences) and the activated GPIIb/IIIa antibody JON/A (#M023-2, Emfret) were added. Washed platelets were activated with 0.5 μg/mL botrocetin (#V5625, Sigma) and 10 μg/mL human von Willebrand factor (#HCVWF-0190, Haematologic Technologies Inc.). For platelet aggregation, whole mouse blood was centrifuged for 15 minutes at 1000 rpm, platelet rich plasma (PRP) was incubated in a 1:100 dilution of CD9-APC or CD9-PE (Abcam) for 15 minutes after samples were washed and resuspended in plasma to a concentration of 50 × 10⁶ platelets/mL. Labeled platelets were mixed 1:1 and agonist-stimulated at 37°C while shaking similar to published by others [21 (link)].

Platelets or bone marrow single cell suspensions were stained for flow cytometry analysis as previously described [30 (link), 31 (link)]. Antibodies used were: CD61-FITC, CD41-PE, Sca1-PECy7, CD34-FITC, Lin-cocktail-APC, CD16/CD32-PE, cKit-PerCP, CD71-FITC, Ter119-V405 (BD Pharmingen, Oxford, United Kingdom), Clec2–FITC (AbD Serotec, Kidlington, United Kingdom), CD42a-FITC, CD42b-DL649, CD42c-FITC (Emfret, Wurzburg, Germany), GPVI-PE (R&D, Abingdon, United Kingdom), CD9-PE, CD31-PECy7 (Abcam, Cambridge United Kingdom) and CD49b-PB (BioLegend, San Diego, CA).