Fitc labelled anti human fibrinogen antibodies

The human isolated platelets or PRP were incubated with different concentrations of 1,8-cineole or a vehicle control for 5 min in the presence of FITC-labelled anti-human fibrinogen antibodies (Dako, Thetford, UK) and PECy5-labelled CD62P (P-selectin) antibodies (BD Biosciences, Berkshire, UK). Platelets were then activated with CRP-XL (0.5 µg/mL), ADP (2.5 µM using PRP) or thrombin (0.025 U/mL using isolated platelets) for 20 min at room temperature. Following this, 0.2% (v/v) formyl saline was added to fix the platelets and the levels of fibrinogen binding (a marker for inside-out signalling to integrin αIIbβ3) and P-selectin exposure (a marker for α-granule secretion) were measured by flow cytometry (Accuri C6, BD Biosciences, Berkshire, UK). The median fluorescence intensity was used to assess the levels of fibrinogen binding and P-selectin exposure on the platelet surface. The level of fluorescence obtained with the vehicle control was taken as 100% to calculate the levels of fibrinogen binding and P-selectin exposure in 1,8-cineole treated samples.

Fibrinogen binding (a marker for platelet inside-out signalling to integrin αIIbβ3) and P-selectin exposure (a marker for α-granule secretion) were measured by flow cytometry (Accuri C6, BD Bioscences, UK). The platelets (PRP) were treated with a vehicle control [0.1% (v/v) DMSO] or with different concentrations of chrysin and its synthetic derivatives prior to activation with CRP-XL (0.5 µg/mL). The levels of fibrinogen binding and P-selectin exposure were measured using FITC-labelled anti-human fibrinogen antibodies (Dako, UK) and PECy5-labelled CD62P antibodies (BD Biosciences, UK), respectively. The median fluorescence intensity was used to assess the levels of fibrinogen binding and P-selectin exposure on the platelet surface. The level of fluorescence obtained with the vehicle control was taken as 100% when compared with the treated samples.