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Cell-cycle analysis

An increase in cell motility can also be a measure of an angiogenic response, as angiogenic factors are known to stimulate cell movement. This is observed in the Boyden chamber assay but is more accurately measured using phagokinetic track assay methods. This uses colloidal gold-plated coverslips to serve as a substrate for the movement of cells, which displace the colloidal gold leaving a track that can be measured for directional properties and total area This assay has been modified to permit large-scale screening using beads attached to the bottom of 96-well plates. Endothelial cells settle on the bead monolayer and generate tracks similar to those produced in colloidal gold . However, the main limitation of these assays is that cells are migrating on an ‘alien’ substrate and not one present in vivo. Similarly, only a small number of endothelial cells are studied in each case, and the assays are time consuming to analyse and interpret.

An alternative type of migration assay is based on the idea that endothelial cell migration into a denuded area is a pivotal event in wound healing in vivo. Endothelial cell monolayers are prepared and permitted to reach confluence. Using a scraping tool, a portion of the monolayer is then cleared of endothelial cells, providing a margin from which endothelial cells migrate to fill the denuded region. The rate and extent of endothelial cell migration is then monitored microscopically As the confluent endothelial monolayer has been ‘wounded’ by scraping and the endothelial cells are migrating back to re-form the monolayer, this assay is considered to represent one aspect of wound healing. Quantification is arbitrary, and problems are found with requirements for the running of control and experimental groups under identical growth conditions of confluence, and the denuded area must be precise