Figure 5: Characterisation of platelet morphology in samples collected at the exit of the microfluidic device and in the control sample.

(a) Two-color flow cytometry analysis of platelets receptors, indicating the population of CD41⁺/CD42b⁺ platelets. (b) Single colour flow cytometry analysis of platelet receptor density, indicating the number of CD61, CD42b and CD49b receptors on the surface of platelets (n = 3). (c) Indirect immunofluorescence labelling with an anti--tubulin antibody, revealed by a secondary AlexaFluor488 goat anti-mouse antibody and AlexaFluor546 phalloidin for F-actin staining is performed in the absence (top panel) or presence (bottom panel) of thrombin. Circular tubulin staining, characteristic of unactivated platelets is seen in the samples collected at the exit of the fluidic device (top left), whereas larger fragments without circular tubulin staining are recovered in samples collected from the control (top right). Actin stress fibres (small arrow, full arrow indicate a filopod and dotted arrow a lamellipod) characteristic of activated platelets are seen in the samples collected at the exit of the fluidic device (bottom left), whereas larger elements without organised stress fibres staining are recovered in samples collected from the control (bottom right). Platelets are adherent to fibrinogen. Scale bar 5 m. (d) Electron microscopy observations of platelets on BSA or fibrinogen coated surface, upon TRAP activation. Discoid round platelets are indicated with full white arrows and pseudopods with dotted arrows. Scale bar 5 m.