Journal: Platelets

Article Title: Assessment of Neonatal, Cord, and Adult Platelet Granule Trafficking and Secretion

doi: 10.1080/09537104.2019.1573314

Figure Lengend Snippet: (A) Representative flow cytometry forward (FSC), side scatter (SSC), P-selectin (APC CD62P) and eFluor CD31 fluorescence intensity of resting and TRAP-6-activated adult, neonatal peripheral, and cord blood. Representative fluorescence intensity of resting and activated platelets are in response to HEPES/Tyrode buffer and TRAP-6, respectively. (B) P-selectin expression in adults, neonatal, and cord blood in response to vehicle or ADP, TRAP-6, protease-activated receptor 4 agonist (AYPGKF), or a combination of agonists. (C) P-selectin expression in platelet-rich plasma (PRP) and whole blood in response to high shear stress. PRP and whole blood suspensions were exposed to high shear stresses of 70 dynes/cm2 for 4 minutes. Data are represented as mean ± SEM of at least 3 independent experiments; *p<0.05 compared to adult vehicle; †P<0.05 compared to cord vehicle; □p<0.05 adults vs. neonatal/cord blood.

Article Snippet: Briefly, adult, neonatal, and cord citrated whole blood was diluted with modified HEPES Tyrode buffer prior to incubation with select agonists (10 μM ADP, 10 μM TRAP-6, 200 μM AYPGKF) or vehicle (HEPES/Tyrode) for 30 seconds at 37°C under orbital shaking in a white, flat bottom 96-well plate (Corning Costar, Tewksbury, MA, USA).

Techniques: Flow Cytometry, Fluorescence, Expressing

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