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Measures migration in response to a gradient

Assays for endothelial cell migration

Endothelial cells move, via a process called chemotaxis, along a gradient of angiogenesis-inducing factor(s) such as VEGF. Modified Boyden chamber assays have been used to assess endothelial cell migration. In these assays, endothelial cells are plated on top of a filter (usually coated with a matrix protein such as fibronectin or collagen) and migrate across this in response to a test angiogenic factor placed in the lower chamber. A major advantage of this assay system is the high sensitivity to small differences in concentration gradients The results shown indemonstrate the increased migration of HuDMECs across a filter in response to basic fibroblast growth factor (bFGF) and VEGF. Major disadvantages are the technical difficulty of setting up the assay, problems in maintaining transfilter gradients for prolonged periods (as this equilibrates between wells over time) and difficulty in obtaining accurate cell counts when small numbers of cells traverse the filter. Different methods of counting have been employed. Computers that scan each well of the membrane systematically and software that recognizes haematoxylin and eosin-stained cells produce a measure of cell number per well which has been used to complement simple approaches such as counting the number of cells per field of view. However, both have limitations, for example, the computer software cannot always distinguish between the pores in the membrane and the stained nuclei of the cells. Thus, the operator has to ensure correct identification of each area identified by the computer, which is time consuming. Similarly, counting by eye can cause problems as the entire well is not counted, and there may be differences in the area of the well that is analysed. Sufficient counts are needed to ensure accuracy of the number obtained, which is time consuming but reduces the subjectivity of this method of quantification. Strict criteria should be established and inter- and intraobserver errors employed.