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
M. Koenigsmann et al., 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, Onkologie, 17(5), 1994, pp. 528-537
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.