Under agarose assay

Fig. 3.3. The under agarose assay. (A) The illustration shows how to make a template in an agarose plate, that allows testing of six different combinations of chemoattractants. (B) The expected results from the under agarose assay the extension of migration of the cells from the central well can be quantitated as for the droplet assay by using a calibrated grid under the microscope.

Under agarose assay Migration occurs between an agarose gel and the plastic surface that supports it... 

Figure 1 Three-dimensional migration assays. (A) Under agarose assay. Cells migrating under a sheet of agarose. (B) Confiner assay. Cells migrating between two glass slides. The spacing between glass slides is determined by polydimethylsiloxane (PDMS) micropillars.

To establish a gradient of chemoattractant gradient, various assays which include the under agarose assay and Dunn chamber are developed. 

Rupnick, M.A., Stokes, C.L., Williams, S.K., and Lauffenburger, D.A., Quantitative analysis of random motility of human microvessel endothehal cells using a linear under-agarose assay. Lab. /west, 1988,59 363-372. 

Newton-Nash, D.K., Tonellato, R, Swiersz, M., and Abramoff, R, Assessment of chemokinetic behavior of inflammatory lung macrophages in a linear under-agarose assay. J. Leukocyte Biol, 1990,48 297-305. 

Measurement of leukocyte motility and che-motaxis parameters using a quantitative analysis of the under-agarose migration assay (with D. Lauffenburger). Math. BioscL 44, 121-138 (1978). 

Under agarose. This assay was originally developed to study leukocyte migration (Nelson et al., 1975) however, it has also been applied to the study of chemotactic and chemokinetic effects of FGF on endothelial cells (Stokes et al., 1990). Cells are allowed to migrate under an agarose gel in which a chemoattractant (or a control solution) forms a diffusion gradient. The differential migration of cells toward the chemoattractant is taken as a measure of its chemotactic activity. 

Lauffenbuiger, D., Rothman, C. and Zigmond, S. H. (1983). Measurement of leukoc5rte motility and chemotaxis parameters with a linear under-agarose migration assay. J. Immunol. 131, 940-947. 

TranquiUo, R.T., Zigmond, S.H. and Lauffenburger, D.A. (1988). Measurement of the chemotaxis coefficient for human neutrophils in the under-agarose migration assay. Cell Motil. Cytoskeleton 11, 1-15. 

Heit, B. and Kubes, R, Measuring chemotaxis and chemokinesis the under-agarose cell migration assay Science s STKE [electronic resource] signal transduction knowledge environment. 2003, 2003 PL5. 

Figure 4.1 The comet assay. A single-cell suspension is embedded in agarose on a slide. Cells are then subject to lysis followed by electrophoresis. If present, damaged DNA migrates out of the nucleoid structure during electrophoresis to producing a characteristic comet shape. Double-strand breaks are revealed under neutral conditions, whereas alkali conditions additionally show single-strand breaks and alkali labile sites. Image analysis of stained DNA is used to quantitate the amount of damaged DNA in the comet tail.

This test, called the Comet Assay or single-cell gel-electrophoresis assay, allows the degree of DNA damage to be determined within a nucleic cell population. The principle of the method is based on the microelectrophoresis of nuclei of isolated cells, under basic conditions, on agarose gel (the whole being observed under a fluorescence microscope). 

Visualize and photograph the agarose gel stained with ethidium bromide under transillumination at 300 nm (UV light). The nucleic acid-lipoplex should remain inside the well, while the free or weakly bound nucleic acid should run in the gel. An example of gel retardation assay is shown in Fig. 2. 

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