QUANTIFYING CELL-ADHESION OF THE COLON-CANCER CELL-LINE HT-29 TO EXTRACELLULAR-MATRIX PROTEINS - COMPARATIVE-EVALUATION OF AN ISOTOPIC, A FLUOROMETRIC, AND A COLORIMETRIC METHOD

Background: Cell adhesion to extracellular matrix (ECM) components can be analyzed by quantifying the fraction of adherent cells. Various in direct methods for cell counting are being used for this purpose. Howe ver, difficulties arise if results are to be reproduced and compared b etween different methods or laboratories. We therefore evaluated compa ratively three indirect cell counting methods with respect to reproduc ibility, accuracy validity, and practicability. Material and Methods: HT 29 colon adenocarcinoma cells were labeled with either the isotope Cr-51 or the fluorochrome methyl-umbelliferyl-heptanoate or by protein staining with amido black 10B. Signal intensities of the cells incuba ted on or adhering to extracellular matrix proteins were measured eith er by gamma counting, fluorometry or photometry and correlated to the actual cell numbers. Furthermore, the influence of assay conditions on signal intensities was evaluated. Results: Highly significant linear correlations between total cell numbers plated per microtiter well and signal intensity were found with each method, This was also true for the fraction of cells adhering to a variety of ECM proteins. Highest r eproducibility and accuracy was found for the chromium assay, followed by the fluorometric and the colorimetric assays. Signal intensity in the fluorometric assay was affected by the type of matrix protein and the incubation time. Conclusions: While the chromium assay is most acc urate, it cannot be generally favored. since false-high adhesion is me asured on some matrix proteins as shown by comparison with direct cell counting, and also because of environmental reasons and costs. The fl uorometric assay is the most rapid and the colorimetric is the least e xpensive method. In conclusion, each method has its specific advantage s and disadvantages. This analysis provides a basis for choosing the a ppropriate method for studying subtle regulatory changes in cell adhes ion to ECM proteins.