The Chemicon QCM™-Fibronectin Quantitative Cell Migration Assay uses a Boyden chamber assay design to help quantify cell migration parameters. The assay consists of a hollow plastic chamber sealed at one end with a porous membrane. This resides in another chamber, which is usually filled with media or a chemoattractant. Cells are seeded into the tube and are allowed to migrate for a specified period of time through the pores towards the chemoattractant to the other side of the membrane. At the assay endpoint, the inner tube is removed and carefully washed, and any non-migratory cells on the inside of the membrane are carefully scraped away. The migratory cells on the opposite side of the membrane can then be quantified.
The major advantage of this system is its versatility. Once cells have migrated and non-migrating cells scraped away, the remaining cells can be stained with the dye provided and the membranes photographed for visual representations, or individual cells can be counted from representative fields of view. The assay also comes with a dye-dissociation buffer, which can be used to dissociate the dye from the cells for quantification on a plate reader.
In this assay, the membrane has been coated with fibronectin, and therefore it tests the affect of your assay conditions on fibronectin-mediated cell migration. The assay is designed so that each treatment condition tested has individual migration and adhesion controls. The assay is adaptable to a wide range of conditions and migration can be tested by pre-treating the cells before assay seeding and/or adding different chemoattractants in the media of the outer chamber. Each kit can test 6 different samples.
While the pore size in traditional Boyden assays can vary depending on the cells being used, Chemicon have employed pore sizes of 8 um, which they claim is suitable for most cells. It would be advisable to determine whether your cells are suited to this pore size.
One of the most difficult and time consuming aspects of this assay is scrapping the non-migratory cells from the inside surface of the membrane. The membrane is very thin and therefore easily ruptured. However, inadequate removal of cells on the inner surface results in high background readings. Patience and extreme care will yield accurate results. Other products are available that are easier to quantify and do not require the removal of non-migratory cells. However, I have not tested these products nor have I found any that are also fibronectin coated.
Although the dye provided works well for visualization purposes, care needs to be taken in the removal of excess unbound dye prior to quantification, as this may require numerous wash steps which may dislodge attached cells. However, given the large amount of contaminant DNA from cells lysed on the seeding-side of the membrane, other methods of quantifying cell number based on DNA quantification are not desirable alternatives as they are likely to give a large amount of background staining.