Journal
NATURE PROTOCOLS
Volume 1, Issue 4, Pages 1840-1851Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2006.252
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Funding
- NCI NIH HHS [1R01CA112314-01A1] Funding Source: Medline
- NATIONAL CANCER INSTITUTE [R01CA112314] Funding Source: NIH RePORTER
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The N-acetyl-D-mannosamine (ManNAc) analog Ac(5)ManNTGc, 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 Ac(5)ManNTGc 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.
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