Novocyte 3000

Flow cytometry was done on a BD FACS Calibur, BD LSRII or NovoCyte 3000, and data were analyzed using FlowJo or NovoExpress.

Platelet-leukocyte aggregate analyses were performed in citrated blood stimulated for 5 min with Adenosine diphosphate (ADP). Then, after the lyses of red blood by FACS lysing solution (BD Biosciences, San Jose, CA, USA), samples were centrifuged, stained with anti-CD45 and anti-CD-61 (BD Biosciences, San Jose, CA, USA), and analysed by flow FACS “Novocyte 3000”. A minimum of 10,000 events was collected in the CD45⁺ gate [24 (link)].
Platelet activation was assessed in PRP using an anti-JON/A (Emfret Analytics, Elbestadt, Germany) or anti-CD62P (P-selectin; BD Biosciences, San Jose, CA, USA) antibody. Twenty-five µL of PRP were mixed with a saturating concentration of antibody and the mixture reacted with different concentrations of ADP or Thrombin Receptor Agonist Peptide (TRAP) for 15 min at room temperature. The reaction was stopped by 500 µL ice-cold PBS, and samples were analysed within 30 min. A minimum of 10,000 events was collected [25 (link)].