Intact cell adhesion to glycan microarrays.
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 nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-microL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, 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 nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing 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.
Nimrichter
Leonardo
L
Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
Gargir
Ari
A
Gortler
Monica
M
Altstock
Rom T
RT
Shtevi
Avi
A
Weisshaus
Oori
O
Fire
Ella
E
Dotan
Nir
N
Schnaar
Ronald L
RL
D000818:Animals; D015704:CD4 Antigens / Q000097:blood / Q000276:immunology; D002448:Cell Adhesion; D002645:Chickens; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D011134:Polysaccharides / Q000502:physiology*; D013601:T-Lymphocytes / Q000502:physiology
D015704:CD4 Antigens; D011134:Polysaccharides