Background: Cell adhesion to extracellular matrix (ECM) components can be analyzed by quantifying the fraction of adherent cells. Various indirect methods for cell counting are being used for this purpose. However, difficulties arise if results are to be reproduced and compared between different methods or laboratories. We therefore evaluated comparatively three indirect cell counting methods with respect to reproducibility, accuracy, validity, and practicability. Material and Methods: HT 29 colon adenocarcinoma cells were labeled with either the isotope 51Cr or the fluorochrome methyl-umbelliferyl-heptanoate or by protein staining with amido black 10B. Signal intensities of the cells incubated on or adhering to extracellular matrix proteins were measured either by gamma counting, fluoro-metry 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 reproducibility 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 accurate, it cannot be generally favored, since false-high adhesion is measured on some matrix proteins as shown by comparison with direct cell counting, and also because of environmental reasons and costs. The fluorometric assay is the most rapid and the colorimetric is the least expensive method. In conclusion, each method has its specific advantages and disadvantages. This analysis provides a basis for choosing the appropriate method for studying subtle regulatory changes in cell adhesion to ECM proteins.

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