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Glycobiology Advance Access published online on November 24, 2003
Glycobiology, doi:10.1093/glycob/cwh022
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Submitted on September 9, 2003
Revised on October 2, 2003
Accepted on October 22, 2003

© 2003 Oxford University Press

ORIGINAL ARTICLES

Intact cell adhesion to glycan microarrays

Leonardo Nimrichter 1, Ari Gargir 2, Monica Gortler 2, Rom T. Altstock 2, Avi Shtevi 2, Oori Weisshaus 2, Ella Fire 2, Nir Dotan 2, and Ronald L. Schnaar 3*

1 Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, MD 21205; Instituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
2 Glycominds, Ltd., Lod 71291, Israel
3 Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, MD 21205

* To whom correspondence should be addressed. E-mail: schnaar{at}jhu.edu.

Abstract

A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to non-reducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-µl aliquots on the glycosylated spots. After incubating to allow cell adhesion, non-adherent cells were removed by immersing the slide in PBS, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air-liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to non-reducing terminal GlcNAc residues in different linkages and orientations, but not to non-reducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans, and may contribute to the repertoire of tools for the study of glycomics.


lectins, oligosaccharides, glycomics, CD4+/hepatocyte
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