Abstract
Quantifying the ability of a compound to modulate cell migration rate is a crucial part of many studies including those on chemotaxis, wound healing and cancer metastasis. Existing migration assays all have their strengths and weaknesses. The "scratch" assay is the most widely used because it seems appealingly simple and inexpensive. However, the scratch assay has some important limitations, as the tool introducing the "wound" might injure/stress the boundary cells and/or harm underlying matrix coatings, which in both cases will affect cell migration. This described method is a Cell Exclusion Zone Assay, in which cell-free areas are created by growing cells around removable silicone stoppers. Upon appropriate staining with fluorescent dyes and microscopically visualizing the monolayers, the migration rate is then quantified by counting the cells (nuclei) intruding the void area left by the silicone insert. In the current study human small intestine epithelial cells were seeded on a physiological substrate matrix to produce collectively migrating monolayers. Different substrates were tested to determine the optimal surface for enterocyte adherence and migration and morphological changes monitored. Recombinant human epidermal growth factor and osteopontin purified from urine were tested to see if the established migration assay produces accurate and reliable migration data with human small intestine cells. The obtained data accurately confirmed that the two bioactive proteins modulate cellular migration in a dose-dependent manner. The presented assay can likely be converted for use with other adherent cell lines or substrate matrices and allows for high throughput, while cost is kept low and versatility high. Co-staining can be applied in order to assay for cell death, different cell types, cell stress and others allowing intricate analysis of migration rate of mixed populations and correction for cell viability.