Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry.
The N-acetyl-D-mannosamine (ManNAc) analog Ac5ManNTGc, a non-natural metabolic precursor for the sialic acid biosynthetic pathway, can be used to display thiols on the cell surface. Sugar-expressed cell-surface thiols are readily accessible compared to their protein counterparts, making them ideal for exploitation in cell-adhesion and tissue-engineering applications. This report describes a protocol for the incubation of Jurkat (human acute T-cell leukemia) cells with Ac5ManNTGc and the quantitative estimation of the resulting sialic acid displayed thiols by flow cytometry after a reaction with a water-soluble biotin-conjugated maleimide reagent and fluorescein isothiocyanate-conjugated (FITC) avidin staining. These methods, with minimal optimization, are generally also applicable to other human cell lines. The labeling and flow cytometry steps of this protocol can be performed in five to eight hours.
Sampathkumar
Srinivasa-Gopalan
SG
Department of Biomedical Engineering, Johns Hopkins University, 3400 N. Charles Street, Clark Hall Rm 106A, Baltimore, Maryland 21218, USA.
Jones
Mark B
MB
Yarema
Kevin J
KJ
D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006595:Hexosamines / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry
C523679:1,3,4,6-tetra-O-acetyl-2-acetylthioacetamido-2-deoxymannopyranose; D006595:Hexosamines; D009844:Oligosaccharides; D013438:Sulfhydryl Compounds; D019158:N-Acetylneuraminic Acid