Whole blood samples were collected inside a 3.2% sodium citrate tube (Becton Dickinson, Franklin Lakes, NJ, USA) and processed between 1 and 3 hours after blood collection using flow cytometry-based assay as described before.49 (link)
Platelet reactivity was assessed under basal (unstimulated) conditions and after stimulation with adenosine diphosphate (ADP; 1.2 and 125uM; Sigma-Aldrich, Zwijndrecht, The Netherlands), cross-linked collagen-related-peptide (CRP-XL; a kind gift from Prof. Farndale, Cambridge, UK), and co-stimulation between ADP with 50 uM serotonin (Sigma-Aldrich, Zwijndrecht, The Netherlands). Platelet reactivity was defined by median fluorescence intensity (MFI) of α-granule protein P-selectin (Biolegend, San Diego, CA, USA) expression and fibrinogen (DAKO, Santa Clara, CA) binding to the activated integrin αIIbβ3. All data were extracted using Kaluza 2.1 software (Beckman Coulter, France) and normalized against quality controls to ensure measurement stability.
Platelet reactivity was assessed under basal (unstimulated) conditions and after stimulation with adenosine diphosphate (ADP; 1.2 and 125uM; Sigma-Aldrich, Zwijndrecht, The Netherlands), cross-linked collagen-related-peptide (CRP-XL; a kind gift from Prof. Farndale, Cambridge, UK), and co-stimulation between ADP with 50 uM serotonin (Sigma-Aldrich, Zwijndrecht, The Netherlands). Platelet reactivity was defined by median fluorescence intensity (MFI) of α-granule protein P-selectin (Biolegend, San Diego, CA, USA) expression and fibrinogen (DAKO, Santa Clara, CA) binding to the activated integrin αIIbβ3. All data were extracted using Kaluza 2.1 software (Beckman Coulter, France) and normalized against quality controls to ensure measurement stability.
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