Adhesion assay

DeFlaun, M. F., Tanzer, A. S., McAteer, A. L., Marshall, B. Levy, S. B. (1990). Development of an adhesion assay and characterization of an adhesion-deficient mutant of Pseudomonas fluorescent. Applied and Environmental Microbiology, 56, 112—19-... 

Xu, Q., Miyamoto, S., and Lam, K. S. (2004) A novel approach to chemical microarray using ketone-modified macromolecular scaffolds application in micro cell-adhesion assay. Mol. Divers. 8(3), 301-310. 

Thermally responsive polymers, such as poly( V-isopropyl acrylamide) (NI-PAm), have also been studied extensively for applications related to those previously discussed [112], De las Heras et al. described the synthesis and patterning of NIPAm brushes on SAMs and their subsequent performance during temperature-dependent adhesion assays of BSA and Streptococcus mutans (Fig. 7). The authors employed p.CP to pattern features of hydrophobic hexadecanethiol and backfilled the surface with an initiator-functionalized alkanethiol. Polymer brushes were grown via surface-initiated atom transfer radical polymerization (ATRP). FITC-BSA was then... 

In an adhesion assay different areas of a slide with a certain peptide sequence were covered separately with different cell lines. After washing and staining only the WEHI 2312 cells were found to bind to the spotted area of the glass slide. 

Platelets are then washed via centrifugation (1,100 x g for 10 min) and resuspended at 2 x 10s/mL in HEPES-Tyrode buffer, and kept at room temperature for no longer than 4 hours before use in aggregation/adhesion assays. 

I. Ramos Cell Adhesion Assay (aMediated Adiiesion/VCAM-I)... 

II. a4j37-K562 Cell Adhesion Assay (a4f37-Mediated AdhesionNCAM-1)... 

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. 

Interactions between tumor cells and platelets can be analyzed by different methods. The ability of tumor cells to induce platelet aggregation ( tumor cells induced platelet aggregation TCIPA) is evaluated by the turbidometric assay (Menter et al., 1987 Watanabe et al., 1988 Sugimoto et al., 1991 Tang et al., 1993 Belloc et al., 1995). Besides, the ability of tumor cells to bind platelets can be evaluated by the classic adhesion assay (see below). 

In vitro platelet-tumor cell adhesion assay If the ability of tumor cells to induce platelet aggregation has a prominent role in metastasis formation, it could be expected that its inhibition by specific drugs would reduce the amount of metastases produced by tumor cells with a pronounced TCIPA activity. 

Adhesion assay. 100 /tl of the platelet suspension are plated into flat-bottomed plastic microtiter wells and allowed to adhere for 24 h at 4°C. Nonadherent platelets are removed by washing with PBS/1% BSA. 100 p, of this solution are added to each well and incubated for 1 h at 37°C to block free adherent sites on plastic. 

We will describe next the different adhesion assays which include (1) adhesion to whole organ derivatives (2) to vascular and microvascular endothelial cells and (3) to the subendothelial extracellular matrix and its constituents. 

Even though this adhesion assay gives reproducible results on a fairly wide range of pH (between 6.7 and 7.9), bicarbonate-buffered solutions should be avoided if the assay is to be carried out in open air, because of bicarbonate poor buffering quality under such conditions (Butcher et al., 1979). 

In the foUowing sections, we will describe methods to prepare vascular and organ-specific microvascular EC, methods for phenotypic modulation of EC in vitro (organ-specific modulation by subendothelial matrix, and activation by cytokines), and different types of adhesion assay of tumor cells on cultured EC. 

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. 

I-deoxyuridine 0.5-1 /tCi/ml in complete culture medium for 24—48 h (Martin-Padura et al., 1991 Lafrenie et al., 1992a, b Bertomeu et al., 1993 Lafrenie et al., 1994). Na- Cr 100 /xCi/ml, 30 min at 37°C in serum containing medium (Antonia et al., 1989 Bereta et al., 1991 Haq et al., 1992). Gamma-emitters do not need a scintillation cocktail, and so, if the adhesion assay has been carried out on coverslips, these can be directly placed in scintillation vials and read with a gamma counter. If, however, solubilization is required, it can be achieved by incubation with NaOH (0.5-1.0 N). After transfer of the solubilized material in scintillation vials, radioactivity associated with adherent tumor cells is quantitated with a gamma counter. 

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... 

Tumor cell adhesion to ECM components can be measured in vitro to either isolated molecules coated on the dish, or to complex ECMs produced by cells in culture or extracted from organs. We will describe first methods to prepare the different ECM coatings on the dish, and next the methodology for the adhesion assay. 

Tumor cells that have the ability to attach to some defined substrates with the same apparent efficiency, as measured by the adhesion assays described above, may nonetheless adhere to the different substrates not with the same strength. Therefore, even though each substrate appears to be able to support cell adhesion, the tenaciousness characterizing each interaction may be different, and it may have an influence on the metastatic ability of tumor cells (Leung-Tack et al., 1988). 

St. John, J. J., Schroen, D. J. and Cheung, H. T. (1994). An adhesion assay using minimal shear force to remove non-adherent cells. J. Immunol. Meth. 170, 159-166. 

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