Chapter 9



Microbead Assay for Quantification of Neuronal Adhesion Molecule Interaction by Flow Cytometry *

ATTILA TÁRNOK, URSEL NÖHRENBERG, HANS-JÜRGEN VOLLMER AND STEPHAN SCHUHMACHER

List of Contents

1. Introduction

2. Materials

3. Procedure

4. Homophilic Interaction

5. Heterophilic Interaction

6. Examples

7. Troubleshooting

8. Figures

9. References

10. Suppliers

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1. Introduction return

Synaptic connections are established primarily during embryonic and postnatal development by growth of axons along restricted pathways. The aim of neuroembryologists is to characterize molecular and cellular mechanisms involved in the formation of functional connections between neurons of the brain (Reichard and Tomaselli, 1991). Interactions with molecular components in the local environment can promote or inhibit growth in a specific direction. The molecular signals that may influence decisions during extension at choice points include diffusible factors which act as chemoattractants or chemorepellants. They are released by intermediate or final cellular target areas and can have a long-range function similar to released soluble adhesion molecules in the peripheral blood. Other molecules involved in axonal guidance are cell surface and extracellular matrix glycoproteins. They can contain permissive and/or non-permissive activities for axonal extension and have a short range-function.

By molecular analysis of axonal growth several cell surface glycoproteins belonging to the immunoglobulin (Ig) superfamily have been identified. In their extracellular part they contain multiple Ig-like domains which are combined with iterative domains related to the type three repeat (FNIII) of fibronectin. These Ig/FNIII-like proteins of the nervous system can be structurally subdivided into a GPI-Iinked (F11, TAG-1, BIG-1 and BIG-2) subgroup, a transmembrane subgroup (neurofascin, NrCAM(Bravo), NgCAM (G4), L1, CHL1) as well as NCAM and OCAM and as DCC and neogenin (Brümmendorf et al, 1989, 1993; Kuhn et al, 1991; Lagenauer and Lemmon, 1987; Morales et al, 1993; Nörenberg et al, 1992, 1995; Schuhmacher et al, 1997). Another group are the extracellular matrix glycoproteins as Tenascin-R (TN-R, former designated restrictin) and TN-C (Brümmendorf et al, 1989, 1993) (for details see ).

The investigation of mutual interactions between identical adhesion molecules (homophilic interaction) and different molecules (heterophilic interaction) is performed by different methods. The neurite extension assay measures the axon length of nerve cells grown on substrates coated with the molecule of interest (Lagenauer and Lemmon, 1987). Permissive substrates lead to elongated neurites, interference e.g. with inhibiting antibodies might reduce neurite length and indicate essential binding sites on the respective molecule. Another approach is the expression of the molecules on the surface of host cells as e.g. COS cells (Brümmendorf et al, 1989,1993). Adhesion of the cells with each other or with protein coated microbeads would indicate homophilic interaction of the expressed molecule. Both methods are highly sophisticated and time consuming. Analysis with microbeads and flow cytometry accelerates screening of interactions. Microbead assays were used to detect cells expressing low numbers of surface antigens (Brümmendorf et al, 1993; McHugh and Fulwyler, 1993; Rathjen et al, 1991) and were recently applied to detect serum concentrations of antibody (McHugh et al, 1997). We applied the microbead technique for the screening of molecule interactions, the inhibiting effect of site directed monoclonal antibodies and the interaction of deletion variants of adhesion molecules.

2. Materials return

Neuronal adhesion molecules

- Neuronal adhesion molecules from the Ig superfamily (e.g. F11, NrCAM, Ng-CAM, neurofascin and N-CAM/Bravo) were generated and kindly provided by the group of Prof F.G. Rathjen (Max-Delbrück Centrum für Molekulare Medizin). Their isolation from detergent extracts of plasma membrane preparations of adult chicken brains and purification byimmunoaffinity chromatography is detailed elsewhere (Brümmendorf et al, 1993).

- Extracellular matrix glycoproteins (tenascin-R, TN-R; TN-C) were generated and kindly provided by the group of F.G. Rathjen (Max-Delbrück Centrum für Molekulare Medizin). Their purification from urea, EDTA, EGTA extracts of adult chicken brains by immunoaffinity chromatography is detailed elsewhere (Nörenberg et al, 1995; Rathjen et al, 1991).

- Monoclonal antibodies to the adhesion molecules were generated from Balb/c mice or rabbits and purified as detailed (Brümmendorf et al, 1989; Rathjen et al, 1991).

Microbeads and chemicals

- Covalent coupling was done with covaspheres, non-covalent labeling with bioclean microbeads (both Duke Sci Corp., Palo Alto) of 0.5 µM diameter and blue, red or green fluorescence.

- phosphate buffered saline (PBS), bovine serum albumin (BSA), sodium azide (all chemicals from Sigma Chemical, St.Louis, MI).

Equipment

• EPICS 751 cell sorter (Coulter Corp., Hialeah, FL) optical filters: dichroic filter (TY312, Schott, Mainz, Germany), bandpass filters: 400 nm (NAL 400, Schott) and 575 nm (Coulter Corp.). Other supplier: Omega Optical, UK.

• Bench top flow cytometer e.g.FACSCalibur (Becton-Dickinson,Sanjose, CA).

• Software: MDADS data analysis system (Coulter Corp.), data analysis system DAS (Beisker, 1994), CellQuest (Becton-Dickinson), SigmaPlot (SPSS, Arlington, VA).

• Ultrasonic waterbath

3. Procedure return

Protein labeling of microbeads

Adhesion molecules are coupled to microbeads either covalently (covaspheres) or non-covalently (bioclean microbeads) (Kuhn et al, 1991). Three differently stained beads were available for protein coupling: blue beads (excitation maximum: 365nm, emission maximum: 446nm), green beads (ex.: 469nm, em.: 509nm) and red beads (ex.: 541nm, em.: 611nm).

1. Sonicate bead suspension shortly before coating with protein in an ultrasonic waterbath to separate aggregated beads.

2. Incubate 100 µl aliquots in (PBS) with 50 µg protein for 1h at 37 °C or overnight at 4 °C. Control beads are coated with BSA.

3. Transfer beads after incubation into PBS supplemented with BSA and l% sodium azide and store at 4°C until analysis. No detectable dye leakage is found for up to two days of storage.

Aggregation assay

1. For blocking experiments, preincubate protein-coated beads for 15 min with poly- or monoclonal antibodies (mAB) or Fab fragments of monoclonal antibodies at a concentration of 1-5 µg/ml.

2. Sonicate the bead suspension prior to analysis for 5 min. Incubate single color beads or 1:1 mixture of two different colors for 30 to 60 min at room Lemperature. Use for the analysis of heterophilic aggregation blue/red (UV excitation) or green/red (488 nm excitation) bead combinations.

3. Add 10-fold volume of PBS to stop aggregation and store on ice until analysis.

4. Dilute beads to a concentration at which the count rate on the flow cytometer does not exceed 1,000 beads/sec

Flow cytometry

1. Use double distilled water as sheath fluid and clean sample lines and orifice.

2a. UV-excitation (e.g. EPICS751 cell sorter, Coulter): Setup the appropriate filters: blue beads 400 nm bandpass, red beads 575 nm bandpass and tune the laser to 300 nm all line UV emission and at least 50 mW laser power.

2b. 488 nm excitation (bench top flow cytometer): set the filters for green and red beads at 525 nm and 575 nm, respectively. On flow cytometers with fixed optical filters (e.g. FACSCalibur, Becton Dickinson) green and red fluorescence are detected in channel 1 and 2, respectively.

3. Set all acquired parameter (forward scatter and fluorescence) to logarithmic scale and acquire fluorescence area (or integral) values if possible.

4a. Homophilic interactions are measured with blue or green beads. Set the trigger to the fluorescence of the respective bead and use non-adhering e.g. BSA coated (control) beads for calibration. Optimize the optical alignment for singlet beads. The coefficient of Variation (CV) should be below 3 % and at least three peaks should be resolved.

4b. Heterophilic interactions are measured with blue/red or green/red combination. Set the trigger to forward angle light scatter and calibrate first with BSA coated blue or green beads followed by red beads and then with a bead mixture. CVs should be below 6%.

5. Mix gently before acquisition and acquire 100,000 or more events at a count rate 4 bead by linear regression analysis using the median intensity value from the resolved peaks (1-4).

b) Number of beads for each region (event number per region x aggregate size) and the total number of beads measured.

5. The residual data are (1) percentage of aggregated beads and (2) mean aggregate size (total bead number /total event number).

5. Heterophilic interactions return

Beads in mixed aggregates have altered fluorescence intensities compared to non aggregated beads. In particular, blue or green beads that are attached to red beads display decreased fluorescence intensity with increasing number of attached red beads (Figure 2. A, B; Figure 3. B). In the analysis this "fluorescence quenching" is taken into account. Data analysis is performed as exemplified in Figure 2 B and Figure 3.

1. Display data on a two parameter histogram (blue vs red or green vs red),

a) Create gates for red beads with or without attached green beads (g0 and gl respectively in Fig. 2 B).

b) Create gates for blue or green beads with or without one, two three four or more than four attached red beads (G0 to G5 in Fig. 2 B).

2. Display gated data as single parameter histograms (Figure 3 A, B), analyze each histogram as described and calculate the total bead and event number.

3. The residual data are for each bead type (1) percentage of the beads in heterophilic Aggregates and (2) mean Aggregate size of beads in heterophilic Aggregates.

6. Examples return

Homophilic aggregation

Typical examples for homophilic interaction are shown in Figure 1. Beads coated with BSA or F11 formed only few homophilic aggregates (5-8%, mean size: 1.05-1.06 beads) whereas NG-CAM coated beads were mostly in aggregates (98%) forming large aggregates (size: 10.3). The data that were obtained with blue beads (Covaspheres) clearly resolved at least four peaks. The formation of the aggregates was a fast process. With Ng-CAM coated beads already after 10 min of incubation 98% of the beads were in homophilic aggregates. The size of the aggregates stabilized after around 30 min of incubation. These values remained unchanged for at least 6 h of incubation. The results of the bead aggregation assay were consistent with results on the binding properties of these molecules and their biological activity (Brümmendorf et al, 1993; Kuhn et al, 1991).

Heterophilic aggregation

Typical examples for heterophilic interactions are shown in Figure 2. In Figure 2 A CALEB coated green beads were combined with NrCAM coated red beads. Although NrCAM beads formed large homophilic aggregates (mean size: 5.5 beads) CALEB beads hardly formed homophilic aggregates (size: 1.2 beads ) and hardly any heterophilic aggregation occurred (CALEB with NrCAM: 1.0 %, NrCAM with CALEB: 0.5 %). In Figure 2 B beads coated with CALEB (green) and TN-R (red) form large heterophilic aggregates (CALEB with TN-R: 75%, 4.5 beads; TN-R with CALEB: 97 %, 6.0 beads). Note also the shift in green fluorescence intensity when aggregated to red beads (Fig. 2 B, 3 B). For further examples of neuronal adhesion molecule interactions measured with microbeads see Brümmendorf et al. (1989, 1993), Morales et al. (1993), Nörenberg et al. (1995) and Schuhmacher et al (1997). Other examples of the bead assay are the analysis of protein binding sites by antibodies and aggregation assay with restriction fragments of adhesion molecules (Morales et al, 1993; Nöhrenberg et al, 1992, 1995; Brümmendorf et al, 1989, 1993).

Modifications

For aggregation experiments microbeads of different sizes ranging from 0.05 µm to 2.0 µm diameter are available. Although larger beads are easier to measure their aggregate formation is not as marked due to lower surface to mass ratio. Aggregation is most pronounced with the smallest beads but in particularly when forward scatter signals are used to trigger data acquisition the background resulting from sheath and light scattered on the optical systems is unacceptably high.

Analysis of the histograms might also be automated by histogram analysis software (e.g. Beisker, 1994; ModFit, Verity Software House Inc., Topsham; MultiCyle, Phoenix Flow Systems, San Diego, CA). Two parameter histogram analysis could be partially automated using cluster analysis programs (e.g. Attractors, Becton-Dickinson).

7. Troubleshooting return

• High background

High background can result from dirt in the sheath fluid or in the sheath filters or misalignment of the optical system: replace sheath and filters and realign.

• High CV

High CV of the beads can result from misalignment or poor bead quality. Check alignment with different bead charges.

• No signals

Lack of appropriate signals from the beads can result from misalignment of the flow cytometer. Start alignment with beads coated with substrates that form strong homophilic or heterophilic aggregates then continue alignment with singlet beads.

8. Figures return

[pic]

Figure 1. Analysis of homophilic interaction with microbeads.

Peptide coated blue microbeads were measured at UV excitation. Four peaks are clearly resolved and are indicated by the bead numbers. Beads were coated with the indicated peptides as described and measured on a EPICS751 cell sorter (Coulter). Histograms display fluorescence intensities vs event count. The bars in the figure show the regions used to analyze singlet and aggregated beads. The values in the histogram indicate the calculated mean aggregate size in the >4 region.

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[pic]

Figure 2. Analysis of heterophilic interaction with microbeads.

Heterophilic interaction of CALEB (green) and NrCAM (red) coated beads. Graph displays dotplot of original data each data point representing a single event. The background signals are expected in the left bottom corner and are gated by the region Ri. Note the pronounced aggregate formation of NrCAM beads; (B) Interaction of CALEB (green) and TN-R (red coated) beads. Data display as in (B) except that background signals are excluded by gating. The gates g0 and g1 were applied for red beads with and without attached green beads. The region G0 to G5 were applied to selectively gate green beads attached to no or to more then four red beads, respectively. The respective gated histograms are displayed in Figure 2. Note the decrease of the fluorescence intensity of singlet green beads when attached to red beads. The values in parenthesis show the number of red or green beads attached in the respective region (values > 4 are extrapolated).

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[pic]

Figure 3. Analysis of heterophilic interaction using single parameter histograms gated for red (A) or green beads (B) applying the gates shown in Figure 1 C. (A)

TN-R coated red beads attached to no (go) or 6.5 green beads (gl). (B) CALEB coated green beads attached to no (GO) to 8.2 red beads (G5). Note the decrease in fluorescence intensity of singlet beads with increasing number of attached red beads.

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9. References return

Beisker W (1994) A new combined integral-light and slit-scan data analysis system (DAS) for flow cytometry. Comput Methods Prog Biomed 42: 15-26. return

Brümmendorf T, Wolff JM, Frank R, FG Rathjen (1989) Neural cell recognition molecule F 1 1: homology with fibronectin type III and immunoglobulin type C domains. Neuron 2: 1351-1361. return

Brümmendorf T, Hubert M, Treubert U, Leuschner R, Tárnok A, Rathjen FG (1993) The axonal recognition molecule F 1 1 is a multifunctional protein: Specific immunoglobulin-like domains of F 1 1 mediate interactions with NgCAM and restrictin. Neuron 10: 711-727. return

Kuhn TB, Stoeckli ET, Condrau MA, Rathjen FG, Sonderegger P (1991) Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with Ll(G4). J Cell Biol 115: 1113-1126. return

Lagenaur C, Lemmon V (1987) An LI-like molecule, the 8D9 antigen, is a potent substrate for neurite extension. Proc Nat. Acad Sci USA 84: 7753-7757. return

McHugh TM, Fulwyler Mj (1993) Microsphere-based fluorescence immunoassays using flow cytometry instrumentation. In: Bauer KD, Duque RE, Shankey TV (eds) Clinical flow cytometry. Principles and application. Williams & Wilkins, Baltimore, pp 535-544. return

McHugh TM, Viele MK, Chase EC, Recktenwald Dj (1997) The sensitive detection and quantitation of antibody to HCV by using a microsphere-based immunoassay and flow cytometry. Cytometry 29: 106:112. return

Morales G, Hubert M, Brümmendorf T, Treubert U, Tárnok A, Schwarz U, Rathjen FG (1993) Induction of axonal growth by heterophilic interactions between the cell surface recognition proteins Fll and NrCAM/Bravo. Neuron 11: 1113-1122. return

Nöhrenberg U, Wille H, Wolff JM, Frank R, Rathjen FG (1992) The chicken neural extracellular matrix molecule restrictin: similarity with EGF-, fibronectin type II-and fibrinogen-like motifs. Neuron 8: 849-863. return

Nöhrenberg U, Hubert M, Brümmendorf T, Tárnok A, Rathjen FG (1995) Characterization of functional domains of tenascin-R (restrictin) polypeptide. 1 Cell Biol 130:473-484. return

Rathjen FG, Wolff JM, Chiquet-Ehrismann R (1 99 1) Restrictin: a chock neural extracellular matrix protein involved in cell attachment co-purifies with the cell recognition molecule Fll. Development 113: 151-164. return

Reichard LF, Tomaselli KI (1991) Extracellular matrix molecules and their receptors: functions in neural development. Annu Rev Neurosci 14: 531-570. return

Schumacher S, Volkmer Hj, Buck F, Otto A, Tárnok A, Roth S, Rathjen FG (1997) CALEB a neural member of the EGF family of differentiation factors is implicated in neurite formation. J Cell Biol 136: 895-906. return

10. Suppliers return

DUKE SCIENTIFIC CORPORATION (COVASPHERES, BIOCLEAN MICROBEADS), PO Box 50005, Palo AltoCA, 94303, USA (phone + 1-415-4241177; fax + 1-415-4241158). return

DUKE SCIENTIFIC CORPORATION; DISTRIBUTOR FOR EUROPE: DISTRILAB BV, PO Box 130, AC Leusden, 3830, The Netherlands (phone +31-33-4947834; fax +31-33-4948975)

OMEGA OPTICAL (OPTICAL FILTERS, DICHROIC MIRRORS), PO Box 573, 3 Grove Street , BrattleboroVermont, 05302, USA (phone + 1 -802-254-2690; fax + 1 -802-254-3937)

OMEGA OPTICAL; DISTRIBUTOR IN GERMANY: INSTRUMENTS S.A. GMBH, Bretonischer Ring 13, Grasbrunn 1, 85630, Germany (phone +49-89-460205 1; fax +49-89-463197)

OMEGA OPTICAL; DISTRIBUTOR IN UK: GLEN SPECTRA LTD., 2-4 Wigton Gardens, Stanmore, Middlesex, HA7 1BG, England (phone +44-81-2049517; fax +44-81-2045189).return

PHOENIX FLOW SYSTEMS (SOFTWARE PRODUCTS FOR PC: MULTYCYCLE-DNA ANALYSIS PROGRAM), 11575 Sorrento Valley Road, Suite 208, San DiegoCA, 92121, USA (phone + 1-619-453-5095; fax + 1 -619-259 527668). return

SCHOTT GLASWERKE (DICHROIC MIRRORS, OPTICAL FILTERS), Box 2480, Mainz, 55014, Germany (phone +49-61 31-66-0; fax +49-61 31-66 20 00WWW: ). return

SIGMA CHEMICAL COMPANY (PBS, BSA, SODIUM AZIDE), Box 14508, St. LouisMissouri, 63178-9916, USA (phone +I-314-771-5750; fax +I-314-771-5757). return

SPSS FEDERAL SYSTEMS (U.S.) (SIGMA PLOT SPREADSHEET AND GRAPHICS PROGRAM FOR PC), Courthouse Place 2000 North 14th, Suite 320, ArlingtonVA, 22201, USA (phone +l-703-527-6777; fax +703-527-6866; e-mail corinne@). return

VERITY SOFTWARE HOUSE INC. (SOFTWARE PRODUCTS FOR PC AND MACINTOSH: MODFIT- CELL CYCLE ANALYSIS SOFTWARE), PO Box 247; 45A Augusta Road, TopshamMaineUSA (phone + 1-207-7296767; fax + 1 -207-729-5443; e-mail erity@, ). return

Abbreviations

CV coefficient of variation

FN fibronectin

TN tenascin

N-CAM neural cell adhesion molecule

CALEB checken acidic leucine-rich EGF-like domain containing brain protein

PBS phosphate buffered saline

BSA bovine serum albumin

Correspondence to:

Attila Tárnok, University Hospital, Herzzentrum Leipzig GmbH, Pediatric Cardiology, Russenstr. 19, Leipzig, 04289, Germany (phone +49-(0)341-865-2430; fax +49-(0)341-865-1143

e-mail: tarnok@medizin.uni-leipzig.de)

Ursel Nöhrenberg, Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, Berlin, 13092, Germany,

Hans-Jürgen Vollmer, NMI, Markwiesenstrasse 55, Reutlingen, 72770, Germany,

Stephan Schuhmacher, Max-Delbrück Center for Molecular Medicine, Robert-Rössle Str. 10, Berlin, 13092, Germany

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Part of this work was published in:

A. Radbruch (ed.): Flow Cytometry and Cell Sorting. Springer Lab Manual. 2nd edition. Springer Verlag Berlin, Heidelberg, New York 1999; pp. 86 - 97. ISBN 3-540-65630-8.

Reprint with permission of the Springer Verlag Berlin, Heidelberg, New York

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