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cytoskeleton elements demonstrating a major reorganization

“In gel” 3-dimensional collagen culture

Type I rat-tail collagen was purchased from BD Biosciences (Bedford, MA) and gels were formed according to the manufacturer’s recommendations. HUVECs (passage 2) were added to a final concentration of 6 × 105 cells/mL in 0.5 mg/mL collagen solution. One milliliter of this suspension was poured into a 35-mm culture dish, and the gels were allowed to form at 37°C for 40 minutes. The gels were then overlaid with 1 mL regular medium and incubated at 37°C in a humidified atmosphere containing 5% CO2. After 24 hours in these conditions, more than 95% of the HUVECs were organized into capillary-like structures. Digital images were captured using phase contrast optics and a Kappa CF11DSP charge-coupled device camera (Kappa Opto-electronics, Gleichen, Germany).

When indicated, 20 μg/mL anti–c-met antibody H-190 or 20 μg/mL control rabbit IgG were added to the overlaying medium. In the control experiments, the H-190 antibody was depleted of specific activity by incubation for 1 hour at room temperature with an affinity adsorbent containing immobilized c-met (c-met–Sepharose). The affinity adsorbent was prepared by covalent coupling HGF R/Fc chimera to NHS-activated Sepharose 4 (Amersham), according to the manufacturer’s recommendations.