Monolayer adhesion assay

An alternative assay to estimate intercellular cohesion is the monolayer adhesion assay, originally described by Walther et al. (1973) and modified by Tao et al. (1983). Details on this assay can be found in Section 2.3 below. 

When EC form a confluent monolayer, they are ready to be used as a substrate for the adhesion assay. 

Coating of 96 multiwell plates is done with 20 /xg of proteins in 0.2 ml of PBS/well overnight at 4°C. After accurate washing of the wells with serum free medium, 5 x 10 EC in 0.2 ml of DMEM containing 10% heat inactivated FCS were plated per each well, and incubated at 37°C until very dense (usually 3-4 days). Monolayers are then rinsed with DMEM supplemented with 0.4% BSA (BSA, Sigma fraction V, treated with periodic acid to remove glycoprotein impurities) and used for the adhesion assay as described below. 

Quantitation of tumor cell adhesion on EC monolayers is usually done by adding labeled tumor cells for a short period of time (from 0.5 to 4 h), since this type of interaction during the process of tumor metastasis in vivo is supposed to occur soon after the release of malignant cells into the circulation. We will describe below different methods for labeling and detection of tumor cells to be used in the adhesion assay. 

EC monolayers obtained after growth on either collagen-coated glass coverslips, or tissue culture multiwell plates as previously described (Sections 2.3.3.1 and 2.3.3.2), are rinsed several times in serum Iree medium, and then incubated 1 h at 37°C with assay medium (usually serum free culture medium supplemented with 0.5-1% BSA), before they are used as substrates for the adhesion assay. Tumor cells are detached from culture dishes either by EDTA or by trypsin treatment. In the latter case, trypsinized cells should be allowed to recover from the enzyme treatment in their growth medium for 30 min at 37°C. After rinsing in serum-free medium, labeled tumor cells are resuspended at the desired concentration in the assay medium and plated onto the EC monolayer at a cell concentration ranging between 10 and 2 x 10 /cm. Incubation is... 

Several types of assays have been described, which aim at measuring the adhesion strength between test cells and a substrate (which can be either a cell monolayer, or a matrix coating). We will describe one of these assays in more detail, and mention the others for reference. 

Price, E. A., Coombe, D. R. and Murray, J. C. A simple fluorometric assay for quantifying the adhesion of tumor cells to endothelial monolayers. (1995). Clin. Exp. Metastasis 13,155-164. 

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