Fluorescein isothiocyanate fitc conjugated cd42a

Platelet activation was assessed using flow cytometry at 2 and 4 h post-exposure. Whole blood samples were immediately labeled with monoclonal antibodies (mAbs) for flow cytometric analysis: CD14-conjugated phycoerythrin (PE) (DAKO, Glostrup, Denmark) specific for monocytes, fluorescein isothiocyanate (FITC)-conjugated CD42a (Serotec, Oxford, UK) specific for platelets and isotype-matched control (Serotec, Oxford, UK). CD14-FITC and CD40-PE (Serotec, Oxford, UK) were used for analysis of CD40 positive monocytes. After incubation, cells were fixed and lysed with FACS-Lyse solution (Becton Dickinson, Franklin Lakes, New Jersey, USA) and samples were analyzed using Facs Calibur flow cytometer (Becton Dickinson, Franklin Lakes, New Jersey, USA). Platelet-monocyte aggregation and CD40 positivity of monocytes were expressed as percentage of 2000–2500 collected monocytes. In addition, to determine platelet surface expression of P-selectin and CD40-ligand, whole blood was labeled with CD42a (FITC), CD62p (PE) specific for P-selectin, CD154 specific for CD40 Ligand and isotype controls. Cells were analyzed with a Facs Calibur flow cytometer. Expression of P-selectin and CD40L were expressed as percentage of collected number of platelets (7500).

Samples were obtained at baseline, at 2 h immediately prior to the thrombosis study and at 24 h post exposure, and processed according to previously described protocols [59 (link)]. In brief, blood was taken from an ante-cubital vein using a 21-gauge cannula and anti-coagulated with D-phenylalanyl-Lprolyl-L-arginine chloromethylketone (75 μL; Cambridge Biosciences, UK). Samples were not analysed unless venesection achieved rapid and uninterrupted blood flow. Five minutes after sample collection, samples were stained with the following conjugated monoclonal antibodies: phycoerythrin (PE)-conjugated CD14 (Dako, Denmark), PE-conjugated CD62P, and PE-conjugated CD154 (Becton-Dickinson, UK); PE-conjugated CD40, fluorescein isothiocyanate (FITC)-conjugated CD42a, and FITC-conjugated CD14 (Serotec, USA); and appropriate control isotypes. All antibodies were diluted 1:20. Once stained, samples were incubated for 20 min at room temperature to identify P-selectin and CD40L on the platelet surface and CD40 on the monocyte surface. Platelet–monocyte samples were fixed with FACS-Lyse (Becton-Dickinson). Platelet samples were fixed with 1% paraformaldehyde. Samples were analysed within 24 h using a FACScan flow cytometer (Becton-Dickinson). Platelet–monocyte aggregates were defined as monocytes positive for CD14. Data analysis was performed using FlowJo (Treestar, USA).