Star 635 phalloidin

For determination of the time point of attachment, platelets were stained with CellMask DeepRed (649/666 nm, Thermo Fisher Scientific Inc., Waltham, MA, USA). The dye was diluted in Hepes-Tyrode buffer containing BSA to a final concentration of 2.5 mg mL À1 . 1 mL of platelet working solution was prepared by mixing the diluted dye with the isolated platelet solution to a final cell concentration of 2 Â 10 7 cells per mL. The working solution was incubated for 5 min at 37 1C and 5% CO 2 before Prostaglandin E 1 at a final concentration of 2.6 mg mL À1 was added and the platelets were centrifuged at 480 Â g for 5 min at 21 1C. Subsequently, the platelets were re-suspended in Hepes-Tyrode for 5 to 10 min before imaging. Fixed platelets (after 10 min of thrombin stimulation) were stained for actin using Abberior STAR 635 Phalloidin (Abberior, Go ¨ttingen, Germany) on PAA gels. The images were recorded using a 100Â oil immersion objective (Olympus, Hamburg, Germany, NA 1.4).