ECM Cell Adhesion Array Kit (colorimetric) from EMD Millipore

The interaction of cells with the extracellular matrix (ECM) mediates a variety of fundamental elements of cellular metabolism, signaling, physical support, and mobility. Importantly, the adhesive qualities of cancerous cells within tumors are closely associated with their propensity to metastasize to other parts of the body. In simplest terms, the more adhesive a tumor cell is, the less likely it will metastasize. Given this relationship between cellular adhesion and metastasis, it is not surprising that this has become an area of intense investigation.

The compositional organization of the ECM, which may be described as a complex but ordered network of proteins and sugar polymers, is highly location-specific. This means that the ECM in the lung will have a very different arrangement of proteins and polysaccharide structural components from that in the liver. Also, cells derived from different tissues, or from different tumor types, may have very different adhesive qualities against ECM originating from different sites. In an effort to simplify research endeavors in this highly complex arena, Chemicon has designed an easy-to-use assay kit for measuring cellular adhesion to 7 different ECM proteins simultaneously. The ECM Cell Adhesion Array Kit, sold now by Millipore, is available with colorimetric or fluorimetric endpoints. Both kit types measure adhesion to collagens I, II, and IV, fibronectin, laminin, tenascin, and vitronectin. This review focuses on the colorimetric kit.

Aside from the cells to be tested and assorted cell-harvesting reagents, the ECM Cell Adhesion Array Kit has everything needed to ascertain the relative adhesion of multiple cell types to 7 different ECM-associated proteins. The kit includes a 96-well microtiter tray containing the immobilized ECM proteins, cell staining solution, extraction buffer, and assay buffer. The microtiter tray consists of 12 removable strips, each containing all 7 proteins plus a blank (BSA). The strips can be used all at once or just a few at a time depending on the experimental set up. The protocol is very easy: Equal numbers of cells are first pipetted into the wells of the tray, which is then incubated for 1-2 hours in a cell culture incubator. After aspirating the medium and washing the cells gently a few times with assay buffer, the cells are stained for 5 min, washed again, and the extraction buffer added. This solubilizes the cell-associated stain, which can then be quantified spectrophotometrically at 540-570 nm.

Although the instructions are quite detailed, it should be stressed that they are just guidelines. Optimal parameters will depend on the particular cell lines you are working with. I used the kit to compare the adhesion of cells in which I had knocked down a protein known to bind to integrins (hence, increasing adhesion when the protein is absent) to the parental line. For these cells, the recommended quantity of 100,000 to 200,000 cells per well was too high and resulted in saturating absorbance levels that revealed no differences in adhesion. A repeat of the assay using 25,000 cells per well produced excellent results that corroborated increased adhesion with the knockdown cells.