Abstract
Standard adhesion assays measure cell binding either to immobilized ligands or to cell monolayers in flat-well microtiter plates under static conditions. Typically, these test systems require several washing steps to separate adherent from nonadherent cells. Here, we describe an adhesion assay which avoids these washing steps by employing V-bottom 96-well plates. In this assay, fluorescently labeled leukocytes are allowed to adhere to V-well plates coated with soluble ligand for a fixed time. Thereafter, centrifugal force is applied to separate adherent cells from nonadherent cells. Nonadherent cells accumulate in the nadir of the V-shaped wells and are quantified using a fluorometer with a narrow aperture. This simple and reproducible method has been validated with different classes of adhesion molecule families (selectins and integrins) and is adaptable to several other adhesive interactions. The assay format is suitable for screening applications and may also be used for diagnostic testing. The receptor/ligand interaction chosen as an example to describe the assay methodology is the interaction between the integrin lymphocyte function-associated molecule-1 (LFA-1, αLβ2) and intercellular adhesion molecule-1 (ICAM-1).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ley, K., Laudanna, C., Cybulsky, M.I., and Nourshargh, S. (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat. Rev. Immunol. 7, 678–689.
Butler, L.M., McGettrick H.M., and Nash G.B. (2009) Static and dynamic assays of cell adhesion relevant to the vasculature. Methods Mol. Biol. 467, 211–228.
Mobley, J.L., and Shimizu Y. (2001) Measure-ment of cellular adhesion under static conditions. Curr. Protoc. Immunol. Chapter 7: Unit 7.28
Humphries, M.J. (2009) Cell adhesion assays. Methods Mol. Biol. 522, 203–210.
Weetall, M., Hugo, R., Friedman, C., Maida, S., West, S., Wattanasin, S., Bouhel, R., Weitz-Schmidt, G., and Lake, P. (2001) A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates. Anal. Biochem. 293, 277–287.
Kim, M., Carman, C.V., and Springer, T.A. (2003) Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins. Science 301, 1720–5.
Kim, M, Carman, C.V., Yan, W., Salas, A., and Springer, T.A. (2004) The primacy of affinity over clustering in regulation of adhesiveness of the integrin αLβ2. J. Cell. Biol. 167, 1241–1253.
Etzioni, A. (2010) Defects in the leukocyte adhesion cascade. Clinic. Rev. Allergy. Immunol. 38, 54–60.
Evans, R., Patzak, I., Svensson, L., De Filippo, K., Jones, K., McDowall, A., and Hogg, N. (2009) Integrins in immunity. J. Cell Sci. 122, 215–225.
Weitz-Schmidt, G., Welzenbach, K., Dawson, J., and Kallen, J. (2004) Improved lymphocyte function-associated antigen-1 (LFA-1) inhibition by statin derivatives: molecular basis determined by x-ray analysis and monitoring of LFA-1 conformational changes in vitro and ex vivo. J. Biol. Chem. 279, 46764–46771.
Dransfield, I., Cabañas, C., Craig, A., Hogg, N. (1992) Divalent cation regulation of the function of the leukocyte integrin LFA-1. J. Cell Biol. 116, 219–226.
Kallen, J., Welzenbach, K., Ramage, P., Geyl, D., Kriwacki, R., Legge, G., Cottens, S., Weitz-Schmidt, G., and Hommel, U. (1999) Structural basis for LFA-1 inhibition upon lovastatin binding to the CD11a I-domain. J. Mol. Biol. 292, 1–9.
Acknowledgments
The authors would like to thank Dr. Karl Welzenbach, Novartis, for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Weitz-Schmidt, G., Chreng, S. (2011). Cell Adhesion Assays. In: Shimaoka, M. (eds) Integrin and Cell Adhesion Molecules. Methods in Molecular Biology, vol 757. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-166-6_2
Download citation
DOI: https://doi.org/10.1007/978-1-61779-166-6_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-165-9
Online ISBN: 978-1-61779-166-6
eBook Packages: Springer Protocols