Journal:

Article Title: PECAM-1 is a negative regulator of laminin-induced platelet activation

doi: 10.1111/j.1538-7836.2010.03883.x

Figure Lengend Snippet: Washed platelets were resuspended at a concentration of 4 × 108/mL in Tyrodes buffer containing 2 mM MgCl2 and 0.5 mM CaCl2 and added to culture plates that had been pre-coated with 50 μg/mL of laminin 511. Platelets were allowed to settle, bind, and spread at 37°C for the indicated times, and then detergent lysed and subjected to immunoprecipitation analysis using mAbs PECAM-1.3 for human platelets and 390 for murine platelets. Western blots were developed using the anti-phosphotyrosine mAb PY20 (top panels) and anti-PECAM-1 polyclonal antibodies SEW32-34 (human) and M-20 (mouse) to visualize antigen loading. Maximal phosphorylation of PECAM-1 at the 45 minute time point reflects the time needed for platelets to settle, make contact with the immobilized laminin, and activate the platelets. The result shown is representative of three separate experiments.

Article Snippet: 700 μl of washed human or murine platelets (6 × 10 8 /mL) suspended in modified Tyrodes-HEPES buffer containing 2 mM Mg 2+ were seeded onto laminin or collagen-coated Corning cell culture dishes (Corning Inc., Corning, NY) and incubated at 37 °C for the indicated time period.

Techniques: Concentration Assay, Immunoprecipitation, Western Blot