ECM Cell Adhesion Array Kit, Fluorimetric From Chemicon

The Chemicon ECM Cell Adhesion Array Kit is an excellent method for testing cell attachment to a variety of extracellular matrix (ECM) proteins. The kit includes a 96-well plate with each row coated with a different purified human ECM protein: Collagen I, Collagen II, Collagen IV, Fibronectin, Laminin, Tenascin and Vitronectin. The final row is coated with BSA as a negative control. I also used a separate uncoated 96-well plate of my own as an uncoated tissue culture plastic control. Each column on the plate is a separate strip, allowing you to save unused strips for use later.

The assay concept is basic and follows commonly used, time-dependent cell attachment assays. Cells are seeded at an assay-dependent concentration into each of the wells, given a pre-determined period of time to attach to the substrate. I would recommend the test assays as I describe below to determine optimal seeding given that different cells have very different sizes and seeding dynamics. The kit does recommend a range for both the seeding density and attachment time, but the user is advised to do trials within this range to determine the best seeding density and assay time frame for their cells and conditions. After seeding and attachment, the media is removed; the cell layer is carefully washed and the number of attached cells is quantified. For quantification, the kit comes with a cell lysis reagent and CyQuant GR® Dye which emits a strong green-fluorescence when bound to nuclear acids. The cells that have attached to each substrate are then lysed with the lysis reagent, mixed with the CyQuant GR® Dye and the amount of nucleic acid is subsequently quantified on an appropriate plate reader to infer the number of attached cells.

The major benefit of this assay is the 96-well plate and its pre-coated proteins. This saves both the time and money that would be required to create the same system. Unless you are experienced with attaching proteins to tissue culture plastic, you will also probably have increased confidence in the standardized techniques used by Chemicon to attach proteins in this system.

Unfortunately, assays of this nature have an inherent variability given that cells do not attach equally and the vigor with which separate wells are washed is difficult to standardize. This variability is compounded further when the cells are cultured in 96-well plates; I have found that it is difficult to control the liquid turbulence during wash steps in the small wells of 96-well plates. In addition, the smaller well size means the assay is more sensitive to changes in cell numbers between repeats. To overcome this, absolute care must be taken in the wash steps, particular in maintaining the vigor in which separate wells are washed. The manufacturer also recommends ensuring the wells do not dry at any stage of the assay, although I have found that this makes efficient washing more difficult.

It should also be mentioned that there is a temptation to seed higher than the optimal number of cells in order to achieve detectable amounts of DNA and reduce variability. This is unadvisable as it can change the interactions between the cells and substrate and even reduce attachment efficiency. Ideally, you should run trial assays on tissue culture plastic (in larger wells if necessary) to determine optimal seeding densities and attachment time. Trial assays are also useful in determining the optimal quantification method.

While the CyQuant dye is quite good, I have more confidence in dyes like PicoGreen, which only bind double stranded DNA, thus removing the possibility of RNA and other non-genomic nucleic acid contributing to the assay results. Depending on the cell types and assay procedure used, you may find alternative methods to be suitable. Generally, fluorescent based dyes are much better for quantifying DNA than assays that are based on colorimetric stains, as these assays are often further complicated by the dye binding to tissue culture plastic or the attached proteins.

Of course, the 96-well plates can be adapted for use in other assays beyond cell attachment. Cells can be seeded at lower density and grown for longer periods of time to allow assays such as cell growth, viability, differentiation, etc. to be conducted to determine the role these ECM proteins play in that particular phenotype of the cell.