Observation and characterisation of the glycocalyx of viable human

endothelial cells using confocal laser scanning microscopyy

Anna L. Barker,*a Olga Konopatskaya,*c Christopher R. Neal,c Julie V. Macpherson,a

Jacqueline L. Whatmore,b C. Peter Winlove,d Patrick R. Unwina and Angela C. Shoreb

a Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry,UK CV4 7AL. E-mail: a.l.barker@warwick.ac.uk; Fax: þ44 (0) 2476 524112;Tel: þ44 (0) 2476 522187

b Peninsula Medical School, Exeter, UK EX2 5EQc Microvascular Research Laboratories, Department of Physiology, Southwell Street,University of Bristol, Bristol, UK BS2 8EJ. E-mail: O.Konopatskaya@exeter.ac.uk;Fax: þ44 (0) 117 928 8151; Tel: þ44 (0) 117 928 7416

d School of Physics, University of Exeter, Exeter, UK EX4 4QJ

Received1st October 2003, Accepted19th December 2003F|rst published as an AdvanceArticle on theweb 27th January 2004

This paper describes the use of confocal laser scanning microscopy (CLSM) to observe and characterise thefully hydrated glycocalyx of human umbilical vein endothelial cells (HUVECs). Viable HUVECs in primaryculture were studied at room temperature in HEPES-buffered, phenol red- and serum-free CS-C cell culturemedium. A fluorescein isothiocyanate-linked wheat germ agglutinin (WGA-FITC) (2 mg ml�1, 30 min) wasused to detect N-acetylneuraminic (sialic) acid, which is a significant component of the endothelial glycocalyx.Single confocal sections, less than 1.3 mm thick, were collected at intervals of 0.5 mm, scanning through theentire z-axis of a series of cells. Cell-surface associated staining was observed, which enabled the glycocalyxthickness to be deduced as 2.5� 0.5 mm. This dimension is significantly greater than that measured by electronmicroscopy, for glutaraldehyde-fixed cells (0.10� 0.04 mm). The specificity of WGA-FITC staining wasdemonstrated 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 withethidium 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 visualisationand characterisation of the endothelial glycocalyx in viable preparations, revealing a thickness that is an orderof magnitude greater than found in ex situ measurements on fixed cells.

1. Introduction

A single layer of endothelial cells forms a critical barrierbetween the blood and the interstitial fluid of most organs.The luminal surface of the endothelium is exposed directly tothe blood and is covered by an elaborate carbohydrate-con-taining, polyanionic surface coat called the glycocalyx. Itincludes plasmalemmal components such as the glycosylatedectodomains of integral membrane proteins, proteoglycansand glycolipids, as well as adsorbed plasma proteins such asalbumin.1 Many important normal and pathological vascularfunctions, such as capillary permeability,2 receptor-mediatedtranscytosis,3 transendothelial cell migration,4 thrombosis,5

inflammation,6 blood coagulation,6 oxygen transport7 and sen-sing of wall shear stress,8 are dependent on interactions occur-ring at the level of the glycocalyx. Albumin, located within theluminal glycocalyx, has been shown to increase the anioniccharge and volume exclusion of the glycocalyx, resulting inincreased steric and electrostatic exclusion forces that reducevascular permeability.9

Several studies have used electron microscopy to demonstratethe presence of the glycocalyx in the microcirculation.10–12

Conventional electron microscopy requires that cell specimensbe dehydrated during the fixation procedure so that the totalvolume of extracellular material is significantly reduced or col-lapsed and compressed, leading to the distortion of the glyco-calyx architecture. To eliminate this problem, we have chosento develop methodology centred on confocal laser scanningmicroscopy (CLSM) to study the hydrated glycocalyx ofviable, cultured endothelial cells derived from human umbili-cal vein. CLSM is a well established physical method for col-lecting three-dimensional images of cellular structures of livingcells without destroying important functional features of stu-died specimens.13,14 However, there are no reports on in situobservation of living endothelial glycocalyx with CLSM.The glycocalyx layer has been visualised by lectin binding to

living cells. We used a fluorescein isothiocyanate-linked wheatgerm agglutinin (WGA-FITC), which binds selectively to N-acetyl-D-glucosaminyl residues, a constituent of heparan sul-fate, the major saccharide of cell surface proteoglycans15 andto N-acetylneuraminic (sialyl) residues, making a significantcontribution to the surface charge and representing a majorcomponent of the endothelial glycocalyx.16

Stringent concurrent screening for viability of the cells wasundertaken for all experimental conditions used. The CLSManalysis provided evidence of the extensive expression of theglycocalyx in cultured macrovascular endothelial cells inphysiologically relevant conditions.

y Presented at the Biophysical Chemistry Conference 2003, Warwick,UK, July 21–23, 2003.

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DOI:10.1039/b312189e

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2. Materials and methods

2.1 Materials

Unless stated otherwise, materials were obtained from SigmaChemicals (Poole, UK).

2.2 Methods

Human umbilical cords were obtained with full ethicalapproval within 12 h of birth from normal pregnancies.Human umbilical vein endothelial cells (HUVECs) were iso-lated by collagenase (0.3 mg ml�1) digestion17 and culturedin 35 mm diameter Petri dishes in medium 199 (M199) con-taining 20% fetal bovine serum, 0.05 mg ml�1 gentamycin,20 mg ml�1 endothelial growth factor supplement and 90 mgml�1 heparin at 37 �C in 5% CO2/air. The identity of endothe-lial cells in culture was confirmed by the presence of typical‘‘ cobblestone ’’ monolayer morphology (shown by the phasecontrast image, Fig. 1) as well as by the expression of vonWille-brand factor, VCAM, and angiotensin converting enzyme(ACE), as determined by immunocytochemical staining andWestern blotting. After 3 days in primary culture, at 70–80%confluency, the cells were washed twice with phosphate buf-fered saline (PBS), and WGA-FITC (2 mg ml�1), prepared inHEPES-buffered, phenol red- and serum-free CS-C (Sigma,C 1556) cell culture medium, was applied for 30–40 min.

2.2.1 Confocal laser scanning microscopy. Lectin binding(as WGA-FITC binding) was observed with a confocal laserscanning microscope (LSM 510, Axioplan 2, Carl Zeiss, Jena,Germany). All CLSM images (1024� 1024 pixels, 12 bit pixeldepth) were acquired using a water immersion objective lens(Zeiss, Achroplan 63x/0.95NA W) with a 10� tube lens. Toobserve WGA-FITC binding, an argon laser (l ¼ 488 nm)was used in conjunction with a long-pass filter (l ¼ 505 nm).For transmission images a HeNe laser was used (l ¼ 543 nm).For simultaneous assessment of cell viability, WGA-FITC

and ethidium homodimer-1 were excited using Ar laser linesat l ¼ 488 and 514 nm, respectively, and fluorescence emissionwas measured with filter sets, band-pass l ¼ 505–550 nm andlong-pass l ¼ 560 nm, respectively. Black level (backgroundoffset) was adjusted to eliminate autofluorescence fromunstained cells. To achieve the optimum compromise betweenresolution and image intensity, the confocal aperture was set togive an optical slice of <1.3mm (data from Zeiss). Under theseconditions lateral resolution was sub-micron and axialresolution was �0.65 mm.Images were processed using the LSM Image Browser soft-

ware (Zeiss). Average pixel intensities were calculated over the

area corresponding to single cells (typically equivalent to 10–40 000 square pixels) using Scion Image software (ScionCorporation, USA).To discriminate between binding of WGA to sialic acid

residues and binding to N-acetyl-D-glucosaminyl residues, arange of known glycocalyx-digesting enzymes12,18,19 namelyneuraminidase (1 U ml�1 in CS-C, 30–60 min), hyaluronidase(from Streptomyces hyalurolyticus, 50 U ml�1 in CS-C, 30–60min), chondroitinase (1 U ml�1 in CS-C, 60 min) and hepari-tinase (5 U ml�1 in CS-C, 60 min), were added after a standardstaining procedure with WGA-FITC (2 mg ml�1 in CS-C, 30min). All the confocal microscopy analyses were carried outin an air-conditioned room, at 23� 0.5 �C.Cell viability was simultaneously assessed with ethidium

homodimer-1 (2.5 mM), which undergoes enhancement offluorescence upon binding to DNA and RNA, which is onlypossible for cells with damaged plasma membranes.20 Addi-tionally, a colorimetric 3-[4,5]dimethylthiazol-2,5diphenyl-tetrazolium bromide (MTT) viability test was performed oncells growing as primary culture in 96-well plates, under condi-tions identical to those of the confocal analyses.21 The tetra-zolium ring of MTT is cleaved in active mitochondria, andso the reaction occurs only in living cells.

2.2.2 Electron microscopy. HUVECs grown on cover slipswere incubated in the presence or absence of neuraminidase(1 U ml�1) for one hour. Cells on cover slips were then fixedby immersion in freshly prepared solution comprising 2.5%glutaraldehyde and 0.3% ruthenium red in 0.1 M cacodylatebuffer (pH 7.3), using a technique adapted from Hayat.22 Afterprimary fixation, on ice for six hours, cells were washed inthree changes of 0.1 M cacodylate buffer at 4 �C and post-fixedin 1% osmium tetroxide, 0.3% ruthenium red in 0.1 M cacody-late buffer for 30 min at 4 �C. Cells were washed again in threechanges of cacodylate buffer and then distilled water, prior todehydration with ethanol. This was followed by infiltrationand embedding in Araldite resin. Resin surrounding the cover-slips was trimmed and the block placed in liquid nitrogen toenhance cleavage between the glass coverslip and the overlyingresin (which have different thermal contraction rates). Glassfragments were gently removed from the overlying resin con-taining the cells. The cell layer was trimmed and 0.5 mm surveysections were cut and stained with 1% toluidine blue in 1%aqueous borax for light microscopy. Cell rich areas of theblock were trimmed and 50 nm thick sections were cut (perpen-dicular to the culture surface) and stained with 3% aqueousuranyl acetate and Reynolds’ lead citrate solution.23 Digitalmicrographs were taken on a Philips 100CS microscope toshow the disposition of any ruthenium red deposits on thesurface of the HUVECs.

3. Results

3.1 WGA-FITC binding

Cells incubated with 2 mg ml�1 WGA-FITC showed clearlydetectable fluorescence on the cell surface which appeared after5 min and which increased to a maximum intensity afterapproximately 30 min of WGA-FITC incubation. Representa-tive WGA-FITC staining and a corresponding transmissionimage of HUVECs are illustrated in Fig. 2. It can be seen thatstaining occurs heterogeneously over an area corresponding tothat occupied by cells.Confocal microscopy allows a quasi-optical sectioning of

fluorescing cells by eliminating fluorescence from sourcesbelow and above the focal plane. A number of single confocalsections, less than 1.3 mm thick, were collected at intervals of0.5 mm through the entire z-axis of cells, labelled with WGA-FITC for 30 min. A typical set of results is shown in Fig. 3.The shape and intensity of the fluorescent pattern differed

Fig. 1 Phase contrast image of HUVECs culture in a petri dish,magnification � 400.

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as a function of the level of cross-section scanning, from asmall clearly defined area near to the top of the cell, to the dif-fused pericellular matrix of weaker intensity on the bottom,indicating that the staining is likely to be related to the cell sur-face. For the laser intensity and scan time used, no significantphotobleaching was observed. Fig. 4(a) shows a single opticalslice near to the surface of the cell and a re-construction of thez-stack images as orthogonal projections in the x–z and y–zplanes, through a cell, along planes passing through the greenand red lines shown in Fig. 4. From these projections it can beseen that staining occurs in an arc over the cells, indicating sur-face localisation. Measurement of the glycocalyx thickness ofthe four cells visible in Fig. 4(a) from the orthogonal projec-tions gave a value of 2.5� 0.5 mm (n ¼ 4). The range in thethickness of glycocalyx measured for more than 20 cells in fourseparate experiments was 2.0� 1.2 mm.

3.2 Enzyme treatment

Enzyme digestion was used to elucidate the specificity ofWGA-FITC staining, and to obtain information about thetypes of WGA binding sites.

Neuraminidase cleaves the O-glycosidic linkages betweenthe terminal neuraminic (sialic) acids and the subterminalsugars.24 Incubation of WGA-labelled HUVECs with a med-ium containing 1 U ml�1 neuraminidase resulted in progressivereduction of the fluorescence (Fig. 4(b)). Reconstruction of z-axis intensity profile of cells after neuraminidase digestionreflected a clear diminishing of both the surface staining andits thickness (Fig. 4(b)). The variation in intensity of stainingof FITC-WGA without enzyme treatment under similar condi-tions (60 min, same laser settings and number of confocal slicesrecorded) was less than 5% (data not shown). Therefore, wecan be confident that the decrease in the intensity of stainingwith enzyme treatment is not significantly affected by photo-bleaching of the fluorophore or changes in the intensity ofthe laser during the time course of the experiment.Fig. 5(a and b) show WGA-FITC staining before, (a), and

after one hour incubation with neuraminidase, (b). In Fig.5(c) the decrease in staining is illustrated quantitatively along

Fig. 3 Binding of FITC-WGA to HUVECs in different focal planesvisualised by confocal laser scanning microscopy. Serial optical sec-tions were acquired from the top (top left image) to the bottom (bot-tom right image) of the sample in defined steps of 0.5 mm. Note thefluorescence pattern of staining on the cell surface indicating lectinbinding to the glycocalyx. Bar, 20 mm.

Fig. 4 Confocal fluorescence images for FITC-WGA binding toHUVECs before (a), and after (b), digestion with neuraminidase (1U ml�1, 30 min). In each case, a single optical slice near to the cell sur-face is shown and reconstructions of 16 serial optical sections (step-size0.5 mm) to produce orthogonal cross-sections in the x–z and y–z planesof cells, along directions defined by the green and red lines. The posi-tion of the single slice image in the z-stack is shown by the blue lines inthe x–z and y–z orthogonal projections. Bar, 2.5 mm.

Fig. 2 (a) Confocal fluorescence images of endothelial cells stainedwith WGA-FITC. Bar, 50 mm. (b) Corresponding transmission imageof HUVECs shown in (a).

Fig. 5 (a) and (b) Confocal fluorescence images of FITC-WGA bind-ing to HUVECs. In (b), HUVECs were incubated for 60 min withneuraminadase (1 U ml�1). (c) Line profiles of pixel intensity acrossone cell (as illustrated by the yellow line in (a) and (b)) of the control(t ¼ 0 min) and at t ¼ 60 min digestion with neuraminidase. (d) Plotshowing decrease in average intensity as a function of time of digestionwith neuraminidase. The average intensity was calculated for an areaof �210 k square pixels and was normalised by the initial control valueat t ¼ 0 min.

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a line taken through a single cell, as shown in Fig. 5(a) and5(b). The significant effect of neuraminidase treatment on thefluorescence intensity can clearly be seen. Fig. 5(d) shows theaverage intensity over an area of 210 000 square pixels,recorded from images taken during 60 min of neuraminidasetreatment. The reduction in fluorescence intensity demon-strates a smooth trend. Fluorescence was found to diminishby 64� 3% (n ¼ 5) after 30 min and by 78� 2% (n ¼ 2) after60 min.Electron microscopy examination of fixed endothelial cells

stained with ruthenium red indicated the presence of glyco-calyx, visible as a layer of dark material on the apical surface(Fig. 6(a)), with a thickness of 0.10� 0.04 mm (n ¼ 6). Inagreement with the in situ observations reported above, neur-aminidase-treated cells which have been fixed lack any obviousglycocalyx (Fig. 6(b)).The results of fluorescence intensity measurements, after

incubation with a series of enzymes, are summarised inTable 1. The distribution of WGA-FITC signal after hepariti-nase (typical data shown in Fig. 7), hyaluronidase and chon-droitinase ABC revealed relatively low levels of glycocalyxremoval, so that reduction in average intensity was 21� 8%(n ¼ 9), 13� 5% (n ¼ 7) and 29� 3% (n ¼ 9) of the controllevel (100%) after 60 min of heparitinase, chondroitinase andhyaluronidase treatment, respectively.

3.3 Cell viability

The aim of this study was to examine cells in a physiologicallyrelevant environment, where the cells are likely to haveretained normal functional characteristics. Therefore, themaintenance of cell viability was of paramount importance,particularly given the potential cytotoxic effect of laser illumi-nation25 and FITC-labelled lectins.26 Cell viability was testedusing two approaches. Firstly, the preincubation and contin-ued presence of ethidium homodimer-1 (2.5 mM, 20 min),throughout all labelling and digestion procedures, demon-strated that all cells monitored maintained intact plasmamembranes. Secondly, cell viability assessed by MTT revealedno statistically significant differences between control andWGA-FITC, or control and WGA-FITC followed by neur-aminidase-treated cells (P ¼ 1, Mann–Whitney test, threeindependent cell populations, data not shown). These dataconfirmed that HUVEC monolayers retained viabilitythroughout all the procedures of WGA-FITC binding andneuraminidase treatment.

4. Discussion

It is well established that the luminal surface of the endothe-lium is lined with glycocalyx, a network of carbohydrate-richmembrane-bound molecules.1 Electron microscopy studies

have assessed the thickness of the endothelial glycocalyx as lessthan 100 nm.12,27 Recently, developed methods, such as in vivovisualisation by intravital microscopy, provided informationabout structures extending much further from the cell surface,averaging 0.6 mm.28 Although it is still a matter of debate,29 itis possible that collapse of highly hydrated surface structuresduring chemical fixation of specimens for electron microscopypreparation may lead to a considerable underestimation of thereal thickness of the glycocalyx.The present study focused on using CLSM applied to iso-

lated HUVECs, as a model to demonstrate non-invasivelythe in vitro expression of the glycocalyx. By this approach wehave shown that the apical surface of HUVECs is covered withan extensive layer of glycocalyx, with an estimated thicknesson the micron scale. It is worth noting that the CLSM proce-dure involved several washing steps, which may have removedplasma proteins associated with, and contributing to, the for-mation of the endothelial surface layer, which suggests thatthe thickness determined is unlikely to be overestimated. Therelatively small molecular size of WGA (molecular weight 36kDa, volume ca. 64 nm3 30) used as a probe for the dissectionof cell surface associated molecules, rules out the possibilityof it having a significant role in the observed glycocalyx thick-ness. Interestingly, the comparative detailed electron micro-scopy examination with the cationic dye, ruthenium red,demonstrated considerably lower thickness of glycocalyxstructures in HUVECs (0.10� 0.04 mm) consistent with earlierelectron microscopy studies of endothelial cells.12,27 This sug-gests that the preparation and measurement routine for elec-tron microscopy results in significant peturbation of theglycocalyx layer thickness. The much larger dimension of theglycocalyx in HUVECs observed with CLSM may there-fore be considered as a characteristic of the unperturbedmonolayer.In addition to the clear consequences of sample preparation

procedures, the discrepancies in the measurements of the

Fig. 6 Electron micrographs of HUVECs stained with rutheniumred. (a) control, (b) after digestion with neuraminidase (1 U ml�1 60min). The glycocalyx appears in (a) as a dark, electron dense layeron the apical cell surface. Bar, 0.5 mm.

Fig. 7 Confocal fluorescence images of endothelial cells stained withWGA-FITC. (a) control, (b) after digestion with heparitinase (5 Uml�1, 60 min). Bar, 50 mm.

Table 1 Percentage reduction in fluorescence intensity with timeduring incubation with various enzymes. For each enzyme treatment,the amount of enzyme (in U ml�1), duration of incubation (in min)and percentage reduction in fluorescence intensity relative to anuntreated control are shown

Enzyme [enzyme] (U ml�1)

Time/

min

Reduction

in intensity

Neuraminidase 1 30 64� 3% (n ¼ 5)

45 72� 2% (n ¼ 2)

60 78� 2% (n ¼ 2)

Heparitinase 5 60 21� 8% (n ¼ 9)

Chondroitinase ABC 1 60 13� 5% (n ¼ 7)

Hyaluronidase 50 30 11� 4% (n ¼ 9)

45 18� 5% (n ¼ 9)

60 29� 3% (n ¼ 9)

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dimension of the glycocalyx in the present study, and datapublished earlier in vivo,18 might also partly arise from thestructural heterogeneity of the endothelial glycocalyx in var-ious species and vessels. All known glycocalyx estimates havepreviously been carried out on animal blood vessels ratherthan human, and have shown considerable variations in com-position and thickness, which depended on vascular tree, ageof animals, and even the specific region of the vessel.12,31

It would be reasonable to expect the glycocalyx morphology ofcultured human endothelial cells to have its own unique char-acteristics. It is also likely that glycocalyx thickness would beinfluenced by a variety of environmental factors such as den-sity of cells, culture conditions and shear stress. Henry et al.have estimated the glycocalyx thickness in vivo as 0.5 mm,18

which is clearly less than our studies. Many factors could con-tribute to this difference eg, different species, in vivo vs. in vitroconditions. Additionally, the measurements of Henry et al.were made under physiological flow and it is not fully under-stand how this may affect the thickness of the glycocalyx,although it has been shown that flow-induced shear stressenhances glycosaminoglycan synthesis in vascular endothelialcells in vitro.32

WGA recognises sialyl residues, which are reported to beexpressed at high concentration in umbilical endothelium.16

Sialic acids are 9C monosaccharides that link to the terminalgalactose, N-acetylgalactosamine, or other sialic acid residuesin carbohydrate chains that are attached to glycoproteins orglycolipids. The hydrolysis of this linkage by neuraminidaseis used as a common tool to identify the specific presence ofsialic residues on a cell surface.12 This standard procedure ofusing glycocalyx-degrading enzymes to demonstrate the tar-geted sites for WGA binding was considered appropriate tovalidate the new experimental approach. Due to their terminallocation, sialic acids on the cell surface are among the firstmolecules encountered by other cells coming in contact withthe cell.33 In addition, the carboxylate group at C-1 is deproto-nated at physiological pH,34 making sialic acid the only sugarin glycoproteins which bears a net negative charge. Sialic acidshave also been shown to inhibit interactions between mole-cules and contribute to the anti-adhesive nature of luminalendothelium, including HUVECs.34

In the experiments reported herein, neuraminidase removedWGA-FITC labelling effectively, but not completely. The resi-dual labelling could be ascribed to interaction with N-acetyl-glucosamine, a constituent of glucosaminoglycans such asheparan sulfate or hyaluronate, which are both expressed inHUVECs.35 This is supported by the observation of glycocalyxremoval by heparitinase and hyaluronidase, which act on gly-cosidic bonds of N-acetylglucosamine, in heparan sulfate andhyaluronate molecules, respectively. Hyaluronidase alsocaused a marked removal of the fluorescent staining. Interest-ingly, however, the kinetics of the enzyme’s action was differ-ent from that described by Henry et al. in vivo, wherehyaluronidase induced a highly pronounced effect in hamstervasculature, with the maximum achieved after one hour of sys-temic infusion.18 Variation in the properties of the glycocalyxin different species, as well as different incubation tempera-tures, could explain the discrepancy. Low levels of chondroitinsulfate may explain the absence of chondroitinase-induced gly-cocalyx disruption on the cell surface in our study. The lack ofenzymatic effect may also be due to steric hindrance of themolecules in the heterogeneous glycocalyx structure.

5. Conclusions

In conclusion, we have developed a methodology for highresolution CLSM coupled with direct staining with fluores-cently labelled lectin, WGA-FITC, to enable the visualisationof the sialic acid-enriched glycocalyx in viable HUVECs. The

specificity of the WGA-FITC staining was demonstrated bytreatment with several enzymes known to degrade glycocalyx,from which neuraminidase was found to be most effective. Byrecording single confocal sections through the z-axis of cellsthe thickness of the hydrated glycocalyx was determined tobe an order of magnitude greater than that found in ex situmeasurements on fixed cells. Viability was confirmed inthe present studies using double labelling with ethidiumhomodimer-1 as an indicator of plasma membrane integrity,the MTT assay and demonstration of morphologicallyundisturbed monolayers throughout the imaging process.These tests allow us to be confident that the measurementsof glycocalyx relate to viable cell monolayers.To our knowledge, this is the first report using CLSM for

quantitative in situ analysis of the fully-hydrated structurally-intact glycocalyx in human macrovascular endothelium. Thereis scope to now use this technique to further investigate theeffect of fluid-flow generated shear stress on the glycocalyx.

Acknowledgements

We thank the Wellcome Trust for financial support (GR063435).

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