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

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 mug 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 mum thick, were collected at intervals of 0.5 mum, 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 mum. This dimension is significantly greater than that measured by electron microscopy, for glutaraldehyde-fixed cells (0.10+/-0.04 mum). 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-2,5-diphenyltetrazolium 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.