Anti human cd62p

Flow cytometry was used to measure the expression levels of CD62P (P-selectin), GPIIb/IIIa (integrin α_IIbβ₃), CD63 (lysosomal glycoprotein), and phosphatidylserine on perfused platelets.
Following blood perfusion through a flow channel with a stenotic region but with no capture region, a 5 μL aliquot of blood supernatant was incubated for 20 minutes with anti-human CD62P, PAC−1, anti-human CD63, or annexin V (BD Biosciences, San Jose, CA, USA) to label P-selectin, active GPIIb/IIIa, lysosomal glycoprotein, and phosphatidylserine, respectively. Two 5 μL blood aliquots were obtained and labeled prior to perfusion. Platelet activation in one aliquot was achieved by addition of thrombin immediately prior to labeling (c = 0.1 units/mL, EMD Millipore, Billerica, MA, USA) and other aliquot was left unstimulated to serve as positive and negative activation controls, respectively. Blood was not treated with PPACK for thrombin activated positive control. To locate platelets in flow cytometry analysis, another 5 μL blood aliquot was labeled with anti-human CD41b (BD Biosciences, San Jose, CA, USA), which binds to the GPIIb subunit of GPIIb/IIIa regardless of the activation state of the receptor. Following labeling, platelets were fixed in 1% paraformaldehyde and stored at 4 °C. Analysis of 100,000 events was conducted on a FACScanto analyzer (BD Biosciences, San Jose, CA, USA).

Flow cytometry was used to measure the expression levels of CD62P (P-selectin), integrin α_IIbβ₃, CD63 (lysosomal glycoprotein), and phosphatidylserine. Following blood perfusion through an albumin coated flow chamber in the absence of a capture region, a 5 μL aliquot of blood supernatant was collected and incubated for 20 minutes with anti-human CD62P, PAC-1, anti-human CD63, or annexin V (BD Biosciences, San Jose, CA, USA) to label P-selectin, active α_IIbβ₃, lysosomal glycoprotein, or phosphatidylserine, respectively. Two 5 μL blood aliquots were labeled prior to perfusion. One aliquot was stimulated by the addition of thrombin immediately prior to labeling (c = 0.1 units/mL, EMD Millipore, Billerica, MA, USA) and the other was left unstimulated to serve as positive and negative controls, respectively. A 5 μL aliquot was also labeled with anti-human CD41b (BD Biosciences, San Jose, CA, USA), which binds to the α_IIb subunit of integrin α_IIbβ₃ regardless of the activation state of the receptor, to locate platelets during flow cytometry analysis. Following labeling, platelets were fixed in 1 % paraformaldehyde and stored at 4 °C. Analysis of 100,000 events was conducted on a FACScanto analyzer (BD Biosciences, San Jose, CA, USA).