Cd9 apc

Aggregation was determined utilizing flow cytometry as previously described [39, 48] . To this end, platelets were labeled with CD9-APC and CD9-PE monoclonal antibodies (1:100 dilution, Abcam) for 15 minutes at room temperature. Following incubation, differently labeled samples were washed twice, mixed 1:1 and then pre-incubated at 37 o C while shaking at 600 rpm for 10 min. Pre-incubated platelets were activated with thrombin or collagen related peptide at 37 o C while shaking at 1000 rpm. At the indicated time points, samples were fixed by addition of 0.5% paraformaldehyde (Carl Roth, Germany) in phosphatebuffered saline. The fixed samples were measured utilizing a BD FACSCalibur (BD Biosciences, Heidelberg, Germany). For quantification, a quadrant was set in the dot plot of respective channels on non-stimulated platelets. The appearance of double-colored events in the upper right quadrant (Q2) was taken as evidence for aggregation and quantified as percentage of total amount of labeled events (Q1+Q2+Q4) at every time point analyzed.

Aggregation was determined utilizing flow cytometry as previously described [41] . To this end, platelets were labeled with CD9-APC and CD9-PE monoclonal antibodies (1:100 dilution, Abcam) for 15 minutes at room temperature. Following incubation, differently labeled samples were washed twice, mixed 1:1, and incubated in 18 µg/ml afatinib (R&D, Germany) for 30 min at 37 o C while shaking at 600 rpm for 10 minutes. The shaking prevented spontaneous aggregation. Pre-incubated platelets were activated with 0.005 U/ml thrombin or 2 µg/ml CRP at 37 o C while shaking at 1000 rpm. At the indicated time points, samples were fixed by addition of 0.5% paraformaldehyde (Carl Roth, Germany) in phosphate-buffered saline. The fixed samples were measured utilizing a BD FACSCalibur (BD Biosciences, Heidelberg, Germany). For quantification, a quadrant was set in the dot plot of respective channels on non-stimulated platelets. The appearance of double-colored events in the upper right quadrant (Q2) was quantified as percentage of total amount of labeled events (Q1+Q2+Q4) at every time point analyzed.