Intact cell adhesion to glycan microarrays

Leonardo Nimrichter1,3,4, Ari Gargir1,5, Monica Gortler5, Rom T. Altstock5, Avi Shtevi5, Oori Weisshaus5, Ella Fire5, Nir Dotan5 and Ronald L. Schnaar2,3

3 Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205; 4 Instituto de Microbiologia Prof. Paulo de Goes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and 5 Glycominds, Ltd., Lod 71291, Israel

Received on September 9, 2003; revised on October 22, 2003; accepted on October 22, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
A rapid and reproducible method was developed to detect and quantify carbohydrate-mediated cell adhesion to glycans arrayed on glass slides. Monosaccharides and oligosaccharides were covalently attached to glass slides in 1.7-mm-diameter spots (200 spots/slide) separated by a Teflon gasket. Primary chicken hepatocytes, which constitutively express a C-type lectin that binds to nonreducing terminal N-acetylglucosamine residues, were labeled with a fluorescent dye and incubated in 1.3-µL aliquots on the glycosylated spots. After incubating to allow cell adhesion, nonadherent cells were removed by immersing the slide in phosphate buffered saline, inverting, and centrifuging in a sealed custom acrylic chamber so that cells on the derivatized spots were subjected to a uniform and controlled centrifugal detachment force while avoiding an air–liquid interface. After centrifugation, adherent cells were fixed in place and detected by fluorescent imaging. Chicken hepatocytes bound to nonreducing terminal GlcNAc residues in different linkages and orientations but not to nonreducing terminal galactose or N-acetylgalactosamine residues. Addition of soluble GlcNAc (but not Gal) prior to incubation reduced cell adhesion to background levels. Extension of the method to CD4+ human T-cells on a 45-glycan diversity array revealed specific adhesion to the sialyl Lewis x structure. The described method is a robust approach to quantify selective cell adhesion using a wide variety of glycans and may contribute to the repertoire of tools for the study of glycomics.

Key words: CD4+ / glycomics / hepatocyte / lectins / oligosaccharides


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Carbohydrate-mediated cell–cell recognition is emerging as an important component in the repertoire of molecular recognition events that underlie the orderly development and functioning of multicellular organisms (Crocker and Varki, 2001Go; Taylor and Drickamer, 2003Go; Vestweber and Blanks, 1999Go). Lectins (carbohydrate-binding proteins) on one cell surface bind to complementary carbohydrates on an apposing cell to initiate physiologically important cell–cell interactions. Genomic analyses indicate ~100 human lectins, and the diversity of lectin protein folds implies that additional families may yet be discovered (Drickamer and Taylor, 2002Go). Specific carbohydrate–carbohydrate interactions may also mediate cell–cell adhesion (Iwabuchi et al., 1998Go; Pincet et al., 2001Go). A challenge for glycobiology is to develop robust screening methods to identify and quantify the binding specificities of carbohydrate-mediated cell recognition events. The variety and complexity of naturally occurring glycan structures, and the typical multivalent nature of carbohydrate-mediated adhesion (low monovalent site affinity, high polyvalent affinity) add to the complexity of the challenge (Dwek, 1996Go; Lee and Lee, 2000Go).


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Table I. LinearCode abbreviations used in this publication

 
Microarrays have been applied successfully to the quantification of nucleic acid and protein interactions with immobilized complementary binding partners and recently have been extended to the investigation of protein–carbohydrate interactions. Several methods were published in which natural and/or synthetic saccharides were adsorbed or covalently bound to surfaces in a manner suitable for screening binding antibodies and/or soluble lectins (Fazio et al., 2002Go; Fukui et al., 2002Go; Houseman and Mrksich, 2002Go; Schwarz et al., 2003Go; Wang et al., 2002Go). These methods have been heralded as the birth of glycomics, the first steps to large-scale screening of glycan recognition in biological systems (Bidlingmaier and Snyder, 2002Go; Drickamer and Taylor, 2002Go; Flitsch and Ulijn, 2003Go; Hirabayashi, 2003Go; Kiessling and Cairo, 2002Go; Love and Seeberger, 2002Go).

Many of the protein–carbohydrate interactions that occur at cell surfaces are intrinsically multivalent (Lee and Lee, 2000Go). Arrays of lectins (or carbohydrates) on one cell surface interact like Velcro with complementary arrays of carbohydrates on an apposing surface. The best characterized example of the kinetic role of multivalency is binding by the hepatic asialoglycoprotein receptor (Lee et al., 1983Go). On the intact hepatocyte surface, the site affinity of this lectin for lactose is 300 µM, whereas its affinity for a trivalent oligosaccharide with appropriately spaced galactose termini is 7 nM, nearly five orders of magnitude higher. Similarly, carbohydrate–carbohydrate binding may have even lower site affinity, requiring highly multivalent interactions for function (Pincet et al., 2001Go). The off rate of a monovalent carbohydrate-mediated interaction may be so rapid that solid-phase binding to immobilized glycan arrays, which require a wash step to remove unbound protein, may be difficult to detect. This problem has been circumvented to some extent by using multivalent soluble lectins or by generating multivalency using chimeras or secondary binding proteins.

In nature, multivalency is often generated by lectin expression on cell surfaces, where lectin molecules self-associate or cluster in response to multivalent binding arrays on an apposing surface (Weis and Drickamer, 1996Go; Weisz and Schnaar, 1991Go). Here we report methods that detect specific adhesion of intact cells to covalent carbohydrate microarrays engineered on glass slides. These methods take advantage of the natural multivalency of cell surface carbohydrate binding to extend the applicability of glycan microarrays.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glass-slide arrayed carbohydrates
Defined glycosides were covalently arrayed on standard-size glass slides using previously described chemistry (Schwarz et al., 2003Go). The array consisted of 8 rows, 25 columns of 1.7-mm diameter spots separated by a Teflon gasket (Figure 1).



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Fig. 1. Glycan microarray (GlycoChip) glass slide patterned with hydrophobic Teflon mask, creating 200 flat wells that can accommodate up to 1.5 µL of hydrophilic liquid. p-Aminophenyl glycosides were covalently attached to the glass surfaces in the wells via an oligomeric 1,8-diamino-3,6-dioxaoctane linker, as described (Schwarz et al., 2003Go).

 
Adhesion of intact chicken hepatocytes to GlcNAc-terminated glycans
A method for quantifying intact cell adhesion to glass slide glycan arrays was developed and refined using primary chicken hepatocytes, which express the well-defined GlcNAc-specific chicken hepatic lectin on their surface (Drickamer, 1981Go). Initial experiments used slides with multiple spots derivatized with GlcNAc, Gal, linker arm (control), and no modification (Figure 2). Chicken hepatocytes adhered selectively to spots derivatized with GlcNAc glycosides. Cell adhesion to Gal-derivatized spots and control surfaces was very low. Varying the conditions for blocking nonspecific cell adhesion (5 mg/mL or 10 mg/mL bovine serum albumin [BSA]) did not alter the results. Microscopic examination of the wells (Figure 3) confirmed that fluorescence correlated with the adhesion of intact cells to the GlcNAc- but not the Gal- or control-derivatized spots.



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Fig. 2. Selective adhesion of chicken hepatocytes to ß-GlcNAc on a glycan microarray slide. DiIC18-labeled primary chicken hepatocytes (6500 in 1.3 µL) were pipetted onto each spot of a glass slide glycan microarray. Adhesion was quantified by determining fluorescence remaining after removal of nonadherent cells as described in the text. Top: Pseudocolor image of the fluorescence intensity. The spots in each column were uniformly derivatized with ß-galactoside (Gal, columns 2–4 and 6–8), ß-N-acetylglucosaminide (GlcNAc, columns 10–12 and 14–16), or with control linker (control, columns 18–20 and 22–24). Intervening columns (none, columns 1, 5, 9, 13, 17, and 21), which also received aliquots of labeled cells, were underivatized. In the upper four rows, spots were blocked with and cells suspended in medium containing 5 mg/mL BSA; in the lower four rows 10 mg/mL BSA was used. Bottom: Quantification of the fluorescence intensities. Values are given as mean ± SD. Black bars represent spots receiving cells suspended in 5 mg/mL BSA and gray bars represent 10 mg/mL BSA.

 


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Fig. 3. Chicken hepatocyte adhesion to GlcNAc-derivatized spots on a glycan microarray slide. Representative spots of the slide in Figure 2 were viewed under bright field (left column) and epifluorescent microscopy (right column). Individual fluorescent cells are evident. The nonuniform distribution of adherent cells within the well is due to mounding of the 1.3-µL droplet of cells added to each spot.

 
Using slides with a more diverse set of glycans (Figure 4) revealed cell adhesion that was fully consistent with the GlcNAc specificity of the chicken hepatic lectin (Burrows et al., 1997Go). Glycans with a nonreducing terminal GlcNAc residue, whether straight chain or branched, supported robust cell adhesion, whereas glycans with terminal Gal or GalNAc residues supported only background adhesion. Linkage position and anomeric configuration had little effect on GlcNAc-specific cell adhesion. To further test the specificity of the interaction, replicate spots were seeded with cells in control medium or medium supplemented with 25 mM GlcNAc as inhibitor or 25 mM Gal as control. Whereas addition of Gal to the medium did not reduce GlcNAc-specific cell adhesion, addition of GlcNAc reduced adhesion to at or near background levels (Figure 5). These data demonstrate robust glycan-specific adhesion of intact chicken hepatocytes to glycan arrays bearing nonreducing terminal GlcNAc residues.



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Fig. 4. Primary chicken hepatocyte adhesion to diverse glycosides on a glycan microarray slide. DiIC18-labeled primary chicken hepatocytes (6500 in 1.3 µL) were pipetted onto each spot on a glass slide arrayed with a variety of Gal-, GlcNAc-, and GalNAc-terminated glycans. Adhesion was quantified by determining fluorescence remaining after removal of nonadherent cells as described in the text. Top: Pseudocolor image of the fluorescence intensity. Columns were uniformly derivatized with (column number): Galß (1), Galß3(GlcNAcß6)GalNAc{alpha} (2), Galß4GlcNAcß (3), GalNAc{alpha} (4), GalNAcß (5), GlcNAc{alpha} (6), GlcNAcß (7), GlcNAcß3GalNAc{alpha} (8), GlcNAcß4GlcNAcß (9), GlcNAcß6GalNAc{alpha} (10) or control linker (11). Bottom: Quantification of the fluorescence intensity. Data are presented as mean ± SD.

 


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Fig. 5. Inhibition of primary chicken hepatocyte adhesion to glycosides on a glycan microarray slide by soluble GlcNAc. DiIC18-labeled primary chicken hepatocytes (6500 in 1.3 µL) were pipetted onto each spot on a glass slide arrayed with a variety of Gal-, GlcNAc-, and GalNAc-terminated glycans as in Figure 4. Adhesion was quantified by determining fluorescence remaining after removal of nonadherent cells as described in the text. Top: Pseudocolor image of the fluorescence intensity. In the columns marked (I) cells were added in the absence of soluble sugars. In the columns marked (II) cells were added in the presence of 25 mM GlcNAc (rows A–D) or 25 mM Gal (rows E–H). Column numbers correspond to different glycosides as described in the legend to Figure 4. Bottom: Quantification of the fluorescence intensity. Black bars represent cells suspended in medium without sugar supplementation, light gray bars with 25 mM Gal, and dark gray bars with 25 mM GlcNAc. Data are presented as mean ± SE.

 
Selective adhesion of human CD4+ T cells to sialyl Lewis x antigen
The described method was used to test human CD4+ T-cell adhesion to a panel of 45 different glycans (Figure 6). CD4+ T-cells from seven healthy individuals were compared for their adhesive specificities. Robust adhesion of CD4+ cells to sialyl Lewis x antigen (Neu5Ac{alpha}3Galß4(Fuc{alpha}3)GlcNAcß) was detected, perhaps via cell surface L-selectin (Camerini et al., 1989Go). In contrast, the nonfucosylated form of the same glycan (Neu5Ac{alpha}3Galß4GlcNAcß) supported little cell adhesion (p < 0.01, t-test for the sialylated versus nonsialylated structure). Three other glycans (Manß4Glcß, Man{alpha}3Man{alpha}, GlcNAcß4GlcNAcß) supported less robust CD4+ T-cell adhesion that was nonetheless significantly above background.



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Fig. 6. Adhesion of human CD4+ T-cells to diverse structures on a glycan microarray slide. (A) CD4+ T-cells isolated from a single individual were pipetted (6000 cells/spot) onto quadruplicate wells derivatized with diverse glycosides. After incubation to allow cell adhesion, nonadherent cells were removed by centrifugation as described in the text. Adherent cells were fixed and fluorescently stained with propidium iodide. A fluorescent scanner image of the slide is shown, along with a magnified insert demonstrating specific adhesion to sialyl Lewis x (NNa3Ab4(Fa3)GNb, lane 5) and mannose-based structures Mb4Gb (lane 1) and Ma3 Ma (lane 4). Surfaces derivatized with GNb3ANa (lane 2) and Fa2Ab (lane 3), like most other surfaces tested, did not support adhesion of CD4+ T-cells. See Table I for LinearCode abbreviations. (B) Quantification of adhesion of CD4+ T-cells from seven different individuals to diverse glycans on a microarray slide. Cells from different individuals were pipetted onto duplicate wells of each glycan and cell adhesion determined as described. Each box represents signals from 50% of the population. The black lines in the box represent the mean values. The boundary of the box closest to zero indicates the 25th percentile, and the boundary of the box farthest from zero indicates the 75th percentile. See Table I for LinearCode abbreviations.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Biomolecular arrays are powerful tools for determining intramolecular interactions (Bidlingmaier and Snyder, 2002Go; Mirzabekov and Kolchinsky, 2002Go; Schena et al., 1995Go). DNA microarrays are used routinely to determine gene expression, and protein microarrays have been developed to test protein–protein interactions and protein functions. Recently, diverse sets of glycans were arrayed on plastic microwells and glass slides to extend microarray technology to the study of carbohydrate–protein interactions (Fazio et al., 2002Go; Fukui et al., 2002Go; Houseman and Mrksich, 2002Go; Schwarz et al., forthcoming; Wang et al., 2002Go). These have been used to study the binding of soluble lectins (either naturally soluble or engineered to lack a transmembrane domain) and soluble antibodies.

The particular glass-slide-based glycan arrays used in the current study possess the following important features for cell-adhesion based screening (Schwarz et al., 2003Go): (1) Simple, well-characterized chemistry is used to covalently link the glycans to the support; (2) a linker separates the glycan and the solid surface conferring access to cells; (3) the glycans are well defined and bound to the linker by known anomeric bonds; (4) the saccharides are bound by their reducing ends and are presented to cells with a well-defined orientation; (5) the array is produced with high well-to-well and batch-to-batch reproducibility; and (6) simple albumin blocking (hepatocytes) or no blocking at all (T-cells) results in low nonspecific binding, giving excellent signal-to-noise ratios.

The results reported here extend the application of glycan arrays to the quantification of carbohydrate-mediated cell adhesion. This extension provides several potential advantages over the study of soluble lectin binding and broadens the applicability of glycan arrays: (1) Full-length lectins are expressed in their physiological environment at the cell surface; (2) carbohydrate specificity can be screened on primary cells without purifying and expressing the relevant lectin(s); (3) relatively few cells are required—as few as 6,000 cells per spot (1.2 x 106 cells total per 200-spot slide); (4) the effects of physiological and pathological modulation of cells on their carbohydrate binding specificities can be quantitatively determined; (5) the method can be extended to cultured cells engineered to express full-length cell surface lectins (Collins et al., 2000Go); and (6) both protein–carbohydrate and carbohydrate–carbohydrate interactions are amenable to cell-based microarray studies.

In the current studies, we used both preadhesion (1,1'-dioctadecyl-3,3,3,3'-tetramethyl-indocarbocyanine perchlorate [DiIC18]) and postadhesion (propidium iodide) fluorescent staining to label the cells for detection and quantification of adhesion. The advantage of the later procedure is that cells need not be preincubated with fluorescent label, which may alter their adhesive state. We tested CD4+ human T-cells using both methods (data not shown) and found that preincubation with dye affected binding, in that CD4+ T-cells pre-labeled with DilC18 lost their mannose binding but retained their sialyl Lewis x binding.

The loss of mannose/GlcNAc binding by CD4+ T-cells after preincubation may indicate a lectin of low stability, abundance, and/or affinity. Whereas CD4+ T-cells express L-selectin, a lectin capable of binding sialyl Lewis x (Camerini et al., 1989Go), expression of intrinsic mannose/GlcNAc binding lectins on these cells has not been established. The mannose/GlcNAc-dependent adhesion that was detected, therefore, may represent a novel lectin activity on CD4+ T-cells. However, it may also be due to other leukocytes present as minor contaminants in the affinity-purified CD4+ cell population (~10% of the cell population, see Materials and methods), some of which express mannose/GlcNAc lectins (East and Isacke, 2002Go; McGreal and Gasque, 2002Go). The lectin identity and specificity of this binding activity were not analyzed further.

The limitations of cell-adhesion microarray studies are primarily technical. Intact cells are not amenable to long-term storage, cannot be exposed to extreme conditions or detergents, and are less compatible with robotic and high-throughput liquid manipulation systems. Unlike protein–carbohydrate binding assays, cell adhesion requires carefully controlled detachment forces (Schnaar, 1994Go). As in our previous glycan cell adhesion assays (Collins et al., 2000Go; Schnaar et al., 1989Go; Swank-Hill et al., 1987Go; Yang et al., 1996Go), centrifugal force was used for this purpose, requiring the fabrication of acrylic centrifuge carriers so that the cells on the adhesive surfaces were subjected to a uniform detachment force in a sealed, fluid-filled compartment. Although these limitations present a challenge to the application of cell-adhesion based glycan microarray studies, they are offset by the advantages for specific applications. Low numbers of cells required, low background adhesion, reproducibility, and highly specific glycan-dependent cell adhesion were demonstrated using this approach, establishing that intact cells can be used to probe the specificity of carbohydrate-based cell adhesion in a robust microarray format.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycan microarray slides
Glycan microarray (GlycoChip) slides were custom made by Glycominds (Lod, Israel). Defined glycosides were covalently arrayed using previously described chemistry (Schwarz et al., 2003Go).

Chicken hepatocytes
Chicken hepatocytes were prepared by collagenase perfusion of juvenile chicken livers as described previously (Obrink et al., 1977Go). The resulting cell populations were predominantly single cells and >85% viable by trypan blue exclusion. Cell suspensions were maintained at <=4°C until use.

An aliquot containing ~5 x 106 cells was placed in a 1.5-mL microcentrifuge tube, the cells were collected by centrifugation (15 s, 3000 x g), washed once with 0.53 mM EDTA in calcium-magnesium-free Dulbecco's phosphate buffered saline (PBS), and resuspended in 1 mL of the same solution. After counting, the cells were centrifuged as before and then resuspended at a final concentration of 5 x 106 cells/mL in HEPES buffered Dulbecco's minimal essential medium (DMEM-HEPES) containing 3 µM DiIC18 (Molecular Probes, Eugene, OR). After 1 h at 37°C in the dark with constant gentle agitation, the labeled cells were washed three times with DMEM-HEPES and resuspended at 5 x 106 cells/mL in the same medium supplemented with BSA (5 mg/mL or 10 mg/mL as indicated). In some experiments, cells were suspended in medium containing 25 mM Gal or 25 mM GlcNAc (as indicated) immediately prior to use.

Human CD4+ T-cells
Ten milliliters of peripheral blood were collected from each of seven healthy volunteers into two 10-ml EDTA-Vaccutainers. The blood was incubated for 4 h at ambient temperature. Immediately after incubation, the blood samples were centrifuged (230 x g; 10 min); after the plasma was removed, the upper 2 mL of buffy coat were transferred to a fresh 15-ml test tube. CD4+ cells were then negatively enriched using RosetteSep CD4+ enrichment kit (StemCell Technologies), a procedure that yields 90% CD4+ cells with the remaining 10% representing the other leukocytes in the starting population. An aliquot of cells were diluted 1:10 in Türk solution (0.01% crystal violet and 3% glacial acetic acid) and counted. Cells were further diluted to a density of 5 x 106 cells/ml in RPMI-1640 medium containing 2% fetal bovine serum and incubated overnight in a humidified incubator (37°C, 5% CO2, 95% humidity).

Chicken hepatocyte adhesion to glycan microarrays
Prior to use, glycan array slides were washed with distilled water and air-dried. To reduce nonspecific cell adhesion, an aliquot (1.3 µL) of Dulbecco's PBS containing either 5 mg/mL or 10 mg/mL BSA was placed on each spot, and the slide was incubated in a humid atmosphere (a closed Petri dish with moist filter paper) for 2 h at 37°C. Blocking solution was removed by aspiration and replaced with an aliquot (1.3 µL) of cell suspension (5 x 106 cells/mL). After 30 min at 37°C in a humid atmosphere to allow adhesion to proceed, the slides were gently immersed in a vat of PBS, inverted onto an immersed custom Plexiglas centrifuge carrier (Figure 7), which was then sealed to exclude air. The carrier containing the inverted slides was centrifuged at 800 x g at ambient temperature for 20 min to remove nonadherent cells. The carrier was reimmersed in PBS; the slide was removed while immersed, righted, lifted out of the PBS, and placed gently in a Petri dish containing 2% paraformaldehyde in PBS for 30 min at ambient temperature. The slides were washed by transferring to a Petri dish filled with PBS, sealed under a coverslip, and stored in the dark at 4°C.



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Fig. 7. Custom acrylic adhesion chamber for glycan microarray slide cell adhesion assays. The chamber is a modification of a custom 96-well plate adhesion chamber described previously (Collins et al., 2000Go). An acrylic insert (arrow) consisted of a 12.7 x 8.6 x 0.45 cm acrylic sheet with four evenly spaced rectangular holes each 5.7 x 2.2 cm to accommodate four glass slides. The holes were flanked by thin (0.16 cm thick) acrylic spacers (arrowhead) to keep the slides in place. The insert was placed in the chamber, the chamber was immersed in a vat of PBS; slides with adherent cells were gently immersed (cell side up) in the vat, inverted, and placed face down over the rectangular holes. While still immersed, an acrylic top with an attached foam rubber gasket and holes that line up with tapped holes in the chamber was used to seal the chamber using knurled-head polypropylene screws. The fluid-filled chamber was then transferred to a centrifuge carrier, nonadherent cells were removed by centrifugation at 800 x g, and the chamber was returned to the PBS-filled vat. While immersed the slides were removed, righted, and then gently lifted from the vat and placed in a Petri dish containing paraformaldehyde in PBS to fix adherent cells in place for subsequent fluorescent detection.

 
Fluorescent images of the glycan array slides were captured using a Fujifilm LAS-1000 image analyzer (Fuji Photo Film, Valhalla, NY), and fluorescent pixel density was quantified using Metamorph image analysis software (Universal Imaging, Downingtown, PA). Adherent cells were also viewed by bright field and fluorescence microscopy using a Nikon T2000 epifluorescent microscope fitted with a Sony CCD camera.

Human CD4+ T-cell adhesion to glycan microarrays
Prior to use, the glycan arrays slides were washed with distilled water and air-dried. The adhesion assay was initiated by applying 1.2 µL human CD4+ T-cell suspension (5 x 106 cells/ml) on each well on the slide; cells from each individual were spotted on replicate slides. After an incubation of 60 min in a humidified incubator (37°C, 5% CO2, 95% humidity) the slides were gently immersed in a vat of PBS, inverted onto an immersed custom acrylic centrifuge carrier (Figure 7), which was then sealed to exclude air. The carrier containing the inverted slides was centrifuged at 50 x g at ambient temperature for 2 min to remove nonadherent cells. The carrier was reimmersed in PBS; the slides gently removed while immersed, then placed in a slide rack. The cells were then fixed to the slides by immersion in PBS/3.7% formaldehyde for 60 min at ambient temperature. The slides were then washed three times in distilled water and air-dried. The bound cells were fluorescently labeled postfixation by incubation of the slides in propidium iodide solution (50 mg/mL in PBS) for 15 min. The slides were then washed three times in distilled water and airdried in the dark.

Fluorescent images of the glycan array slides were captured using an Affymetrix 428 laser scanner (Affymetrix, Santa Clara, CA) using an excitation wavelength of 522 nm and an emission wavelength of 665 nm; fluorescent pixel density was quantified using OptiQuant image analysis software (Packard, Boston, MA).


    Acknowledgements
 
We thank Charles Campbell, Barbara O. Rauch, and Saul Roseman, Department of Biology, The Johns Hopkins University, for providing chicken hepatocytes. L.N. was supported by a fellowship from the Committee for Postgraduate Courses in Higher Education (CAPES), Ministry of Education, Federal Government of Brazil. R.L.S. is a member of the Glycomind's scientific advisory board and owns rights to Glycominds stock. The terms of this arrangement are managed by The Johns Hopkins University in accordance with its conflict of interest policies.


    Footnotes
 
1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed; e-mail: schnaar{at}jhu.edu Back


    Abbreviations
 
BSA, bovine serum albumin; DiIC18, 1,1'-dioctadecyl-3,3,3,3'-tetramethyl-indocarbocyanine perchlorate; PBS, phosphate buffered saline. The LinearCode for saccharides is fully described (Banin et al., 2002Go), as indicated in Table I for structures used in the current study.


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 Introduction
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 Discussion
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