Platelet Adhesion Under Shear Flow

Blood was collected from retro-orbital puncture into 40 µM D-phenylalanyl-prolyl-arginyl chloromethyl ketone, 5 U/ml heparin, and 40 U/ml fragmin. Samples of 400 µl were preincubated with DiOC₆ (0.5 µg/ml, Anaspec, Fremont, CA, USA) and fibrinogen AF546 (25 µg/ml, Thermo-Fischer Scientific, Waltham, MA, USA) for 5 min before whole blood perfusion. Coverslips coated with micro-spots of type I collagen (1 µl, 100 µg/ml, Nycomed, Hoofddorp, The Netherlands) were mounted on a transparent, parallel plate flow chamber (50 µm depth, 3 mm width, and 300 mm length). Flow perfusion using a shear rate of 1000 s⁻¹ was performed on all samples^{67 (link)}. Brightfield and fluorescence images were captured every 30 s for 3.5 min using an EVOS microscope, equipped with a 60× objective. Surface area coverage was analyzed using semi-automated scripts operating in Fiji (ImageJ)^{68 (link)}.