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An Image-Based Assay of Endothelial Cell Tube Formation as a Model of Angiogenesis

Introduction

Formation of new blood vessels is fundamental to the development of cancerous cell masses and has therefore been the focus of many drug screening and cell signaling research studies. Blood vessel development is a significant event in the development and growth of solid tumors, and is implicated in wound healing, retinopathy and macular degeneration.

Traditional animal models to quantify the degree of blood vessel formation are being replaced by cell culture assays that are easier to set up, statistically reliable and can be automated in a drug screening laboratory.1,2

These assays rely on the endothelial cells ability to form distinct blood-vessel-like tubules in an extracellular matrix (BD Matrigel Matrix) where they can subsequently be visualized by fluorescence microscopy. Although quantification of the tubules can be performed by manual tracing, this method precludes the use of the assay in unbiased high-throughput applications. Much preferred is the use of an automated confocal imaging system such as the BD Pathway Bioimager with sophisticated image and data analysis algorithms. The assay is designed to work with live cells in order to avoid fixation artifacts such as the disruption of tubules.

Methods

Human Umbilical Vein Endothelial Cells (BD HUVEC-2, Cat. No. 354151) were maintained in EGM-2 (Cambrex, CC-3156). The cells were washed and trypsinized (trypsin- EDTA, Cambrex, CC-5012) then resuspended into basal media (Cambrex, CC-3162) containing 0.1% BSA (Cat. No. 354331) and seeded into BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation plates (Cat. No. 354150) at 20,000 cells/well and incubated for 16 to 18h at 37C (5% CO 2 ). These plates contain an optimized BD Matrigel Matrix which promotes endothelial cell tube formation. Suramin (Calbioche
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