Fig. 1

Immunofluorescence and flow cytometry analyses of resting and stimulated platelets confirm the presence of FPR2/ALX. (a) FPR2/ALX^+/+ and FPR2/ALX^−/− mouse platelet were lysed and analysed by SDS-PAGE. Following blotting of the separated proteins on a PVDF membrane, the primary (rabbit polyclonal anti-FPR2/ALX antibody) and secondary (Cy5™ anti-rabbit IgG antibody) antibodies were used to detect FPR2/ALX. 14-3-3-ζ was included as a loading control. The membrane was scanned using a Typhoon 9400 Variable Mode Imager (GE Healthcare, UK) to visualise the bands. (b) Resting and (c) 5 µM U46619-stimulated platelets were fixed and permeabilised by using 0.2% (V/V) TritonTM x-100 before the addition of anti-FPR2/ALX antibody and Alexa Fluor 488-conjugated Phalloidin. Alexa Fluor 647-labelled anti-rabbit IgG antibodies were added to detect FPR2/ALX. As controls, Alexa Fluor 647-anti-rabbit IgG and Phalloidin were added. Negative controls excluded the non-specific binding. Platelets were then visualised by confocal microscopy, objective (1000x). The images shown are representative of several images taken for three separate donors. Human PRP [resting or CRP-XL (0.5 µg/ml) stimulated] was incubated with anti-FPR2/ALX antibodies and detected using Cy5TM anti-rabbit IgG secondary antibodies and analysed by flow cytometry. (d) The bar chart represents the surface expression of FPR2/ALX in platelets, and its expression increases compared to isotype control. (e) A representative histogram showing the elevation of FPR2/ALX levels in activated platelets (green) compared to resting platelets (black). The red line represents the isotype control. Data represent the mean ± SEM (n = 3) of the median fluorescence intensity values. *P ≤ 0.05 and **P ≤ 0.01 values were as calculated by One-way ANOVA