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Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry

Nature Protocols volume 1, pages 1840–1851 (2006)Cite this article

Abstract

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.

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Figure 1: Metabolic expression of thiols on cell-surface sialic acids.
Figure 2: Metabolic incorporation, quantification and applications for cell-surface displayed, non-natural sialic acids.
Figure 3: Quantification of cell-surface thiols (CSTs) by flow cytometry.
Figure 4: Redox modification of Neu5TGc.
Figure 5: Dose dependency of cell-surface thiol expression and cell growth.
Figure 6: Applications of metabolic sialic acid engineering.
Figure 7: Effect of cell-surface thiol (CST) expression on morphology.

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Acknowledgements

We are grateful to K. Konstantopoulos for flow cytometer access, P. Pawar for technical assistance in FACS experiments, J.D. Gearhart and M. J. Shamblott for the kind gift of hEBD-LVEC cells, T.H. Wang for a kind gift of QD655–streptavidin conjugate, and J.M. McCaffery and E. Perkins of Integrated Imaging Facility (Johns Hopkins University, Department of Biology) for help with confocal microscopy. Funding was provided by the Arnold and Mabel Beckman Foundation, the US National Institutes of Health (1R01CA112314-01A1) and the National Science Foundation (QSB-0425668).

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  1. Department of Biomedical Engineering, Johns Hopkins University, 3400 N. Charles Street, Clark Hall Rm 106A, Baltimore, 21218, Maryland, USA

    Srinivasa-Gopalan Sampathkumar, Mark B Jones & Kevin J Yarema

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Sampathkumar, SG., Jones, M. & Yarema, K. Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry. Nat Protoc 1, 1840–1851 (2006). https://doi.org/10.1038/nprot.2006.252

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