Jump to main content
Jump to site search
PLANNED MAINTENANCE Close the message box

Scheduled maintenance work on Wednesday 27th March 2019 from 11:00 AM to 1:00 PM (GMT).

During this time our website performance may be temporarily affected. We apologise for any inconvenience this might cause and thank you for your patience.

Issue 5, 2004
Previous Article Next Article

Observation and characterisation of the glycocalyx of viable human endothelial cells using confocal laser scanning microscopy

Author affiliations


This paper describes the use of confocal laser scanning microscopy (CLSM) to observe and characterise the fully hydrated glycocalyx of human umbilical vein endothelial cells (HUVECs). Viable HUVECs in primary culture were studied at room temperature in HEPES-buffered, phenol red- and serum-free CS-C cell culture medium. A fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC) (2 μg ml−1, 30 min) was used to detect N-acetylneuraminic (sialic) acid, which is a significant component of the endothelial glycocalyx. Single confocal sections, less than 1.3 μm thick, were collected at intervals of 0.5 μm, scanning through the entire z-axis of a series of cells. Cell-surface associated staining was observed, which enabled the glycocalyx thickness to be deduced as 2.5 ± 0.5 μm. This dimension is significantly greater than that measured by electron microscopy, for glutaraldehyde-fixed cells (0.10 ± 0.04 μm). The specificity of WGA-FITC staining was demonstrated by treatments with several enzymes, known to degrade glycocalyx (heparatinase, chondroitinase, hyaluronidase and neuraminidase), of which neuraminidase (1 U ml−1, 30–60 min) was the most effective, removing up to 78 ± 2% of WGA-FITC binding to HUVECs. Cell viability was assessed simultaneously with ethidium homodimer-1 staining and confirmed by standard colorimetric 3-[4,5]dimethylthiazol-2,5diphenyltetrazolium bromide (MTT) test. CLSM thus provides a useful approach for in situ visualisation and characterisation of the endothelial glycocalyx in viable preparations, revealing a thickness that is an order of magnitude greater than found in ex situ measurements on fixed cells.

Back to tab navigation

Publication details

The article was received on 01 Oct 2003, accepted on 19 Dec 2003 and first published on 27 Jan 2004

Article type: Paper
DOI: 10.1039/B312189E
Citation: Phys. Chem. Chem. Phys., 2004,6, 1006-1011

  •   Request permissions

    Observation and characterisation of the glycocalyx of viable human endothelial cells using confocal laser scanning microscopy

    A. L. Barker, O. Konopatskaya, C. R. Neal, J. V. Macpherson, J. L. Whatmore, C. P. Winlove, P. R. Unwin and A. C. Shore, Phys. Chem. Chem. Phys., 2004, 6, 1006
    DOI: 10.1039/B312189E

Search articles by author