Neutrophil spreading occurs on spatially distinct areas of collagen thrombi. Citrated whole blood was perfused over a collagen (2 mg/mL) matrix for 5 minutes at 1800 s−1 to obtain platelet thrombi. Nonadherent platelets were removed by a further 5-minute perfusion with cell-free unsupplemented Tyrode buffer at 1800 s−1 followed by perfusion of isolated neutrophils (1 × 106/mL) in Tyrode buffer supplemented with 1 mM CaCl2/MgCl2 for 5 minutes at 150 s−1. Neutrophil-thrombus interactions were visualized by differential interference contrast (DIC) microscopy at a high (at the apex of thrombi; image on left) or low (to image the sides of thrombi; image on right) focal plane. These images are from 1 experiment representative of more than 10 independent experiments. Neutrophils in images are identified by asterisks. Arrows indicate the presence of SCIP, particularly prominent around the side of the thrombi. Bar represents 30 μm.
