Fitc conjugated anti cd41 mab

Venous blood was drawn into a 3.2 % sodium citrate blood-collecting tube. Whole blood was diluted fivefold by adding a Ca²⁺-free HEPES buffer (145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.5 mM NaH2PO4, 5 mM glucose, and10mM Hepes/Na; Sigma) within 30 min of collection. Diluted blood samples were incubated for 10 min at room temperature with PC5-conjugated anti-CD45 mAb (BD Bioscience, San Jose, CA) and FITC-conjugated anti-CD41 mAb (Beckman Coulter, Marseille, France) for leukocyte-platelet aggregate analysis. Next, samples were fixed with 1 % paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 10 min at room temperature, followed by a dilution of 4.6-fold with distilled water for red cell lysis and sample dilution. Samples were analyzed by flow cytometry [33 (link)]. For detection of platelet activation, blood samples were centrifuged for 10 min at 800 rpm in preparation for platelet rich plasma (PRP). The PRP were diluted tenfold with PBS and incubated for 20 min at room temperature with an anti PE- conjugated anti CD62p monoclonal antibody (BioLegend, San-Diago, CA) and a FITC- conjugated PAC-1 monoclonal antibody (Becton Dickinson. San Jose, CA), specific to the active conformation of integrin αIIbβ3 in the presence or absence of 10 μM ADP (molab) as a platelet activator. Samples were diluted fivefold with PBS and analyzed by flow cytometry.

Platelets were prepared from blood samples of healthy donors, with the addition of acid citrate dextrose (ACD) as anticoagulant. Platelet-rich plasma (PRP) was preliminary obtained from the whole blood by centrifugation at 150 × g for 15 min at 20°C.
PRP was centrifuged at 900 × g for 10 min at 20°C. Platelet-poor plasma (PPP) was removed and pellets were re-suspended in Tyrode's buffer, containing 10% (v:v) ACD. Then, after washing, pellets were re-suspended in Tyrode's buffer, containing Bovine Serum Albumine (BSA), 3 mg/ml.
Platelet counts were performed by a hemocytometer (Coulter, Beckman Coulter, Brea, California, USA); that leukocyte contamination was <1 leukocyte/10⁷ platelets. The purity of the isolated platelets was confirmed by staining with a fluorescein isothiocyanate (FITC)–conjugated anti-CD41 mAb (Beckman Coulter) and analyzing by flow cytometry (Coulter Epics, Beckman Coulter).