Detection and quantification of glycocalicin in mouse plasma.
Blood (50 μL) was collected in 200 μL heparinized PBS and diluted 1:10 with PBS. Cells and microparticles were removed by centrifugation. (A) Immunoprecipitation from normal mouse plasma with p0p 1-5 and a nonimmune IgG1 (control), followed by immunoblotting with p0p 5-FITC. Bound p0p 5 was detected by HRP-labeled rabbit anti-FITC/ECL. (B) Detection and quantification of glycocalicin in mouse plasma and the supernatants of mouse platelets using a sandwich-type ELISA (p0p 3,4-HRP). Washed platelets (109/mL) or platelet-rich plasma were activated with either PMA (50 ng/mL) or thrombin (thr; 0.2 U/mL) or were incubated without agonist (control) for 15 minutes at RT. Supernatants were prepared by centrifugation at 15,000g for 10 minutes and were tested along with normal mouse plasma and a standard of known GC concentration (23.8 μg/mL) in serial dilutions. See “Materials and methods” for details.
