This work was supported by an NSF/ARRA award (IOS 0920357) and an award from the NIH (5R21MH098463).

1. Zellpräparation (Tag -2 bis 0) <ol><li> Split HEK293-Zellen 1: 5 unter Verwendung von 0,05% Trypsin-EDTA-Lösung und Inkubation in Wachstumsmedium bei 37 ° C mit 5% CO 2, bis 60 bis 80% konfluent (2 - 3 Tage). Für jede Bedingung Kulturzellen in 2 x 100-mm-Schalen. </li></ol> 2. Zell Transfektion (Tag 1) <ol><li> Transfektion von HEK293-Zellen mit Plasmid-Codierung Fc-Fusion mit einem Transfektionsreagenz Lipofectamine wie. Alternative Methoden, die in vergleichbaren Transfektionseffizienzen führen, können ebenfalls verwendet werden. </li><li> Rück transfizierten Zellen für 24 Stunden Inkubator. </li></ol> 3. Zellvermehrung (Tag 2) <ol><li> Vorheiz Growth Medium ohne fötales Rinderserum (-FBS) bis 37 ° C beträgt. </li><li> Spülen Geschirr 2x mit 10 ml Wachstumsmedien -FBS, und geben die Gerichte an die 37 ° C-Inkubator für 1 Stunde. </li><li> Spülen der Kulturen Geschirr noch einmal mit 10 ml Wachstumsmedien -FBS, für insgesamt 3 Waschungen. </li><li> IncuBeize die transfizierten HEK293-Zellen bei 37 ° C für weitere 48 h ohne FBS vor dem Sammeln Medien. </li></ol> 4. Bead Aggregation (Tag 4) <ol><li> Medien von Kulturschalen sammeln, übertragen Sie Medien von jedem Paar von Gerichten zu 50 ml konischen Röhrchen und Spin bei 500 g für 5 min, um Zelltrümmer zu pelletieren. </li><li> Filtermedien 50 ml konischen Röhrchen in ein Zentrifugalfilter mit einer 30 ml Spritze und 0,45 um-Spritzenfilter. </li><li> Spin Zentrifugalfilter bei 4.000 × g und 4 ° C bis zum Volumen der konzentrierten Kulturmedium beträgt etwa 500 ul (etwa 15 min). Wiederholen, bis alle Kulturmedien wurde hinzugefügt und eingeengt. </li><li> Fügen 1,5 ul Protein G magnetischen Kügelchen auf 1 ml eiskaltem Bindungspuffer in einem 1,5 ml Mikrozentrifugenröhrchen für jede Probe, auf ein Magnet und entfernen Puffer. Sofort die geballte Kulturmedien, um das Protein G magnetischen Beads hinzuzufügen. </li><li> Drehen Röhrchen bei 4 ° C für 2 Stunden. </li><li> PlAss Rohre auf einem Magneten und Medien zu entfernen. Waschen schnell Perlen zweimal mit eiskaltem 1 ml Binding Buffer, und dann wieder zu suspendieren Perlen in 300 ul Binding Buffer. </li><li> Split resuspendiert Perlen in 2 Röhren, 150 ul in jedes Rohr, und fügen Sie 1,5 ul 200 mM CaCl 2 oder 200 mM EDTA für die &quot;Calcium&quot; und &quot;kein Kalzium&quot; Bedingungen auf. </li><li> Transfer 100 ul von jedem Zustand in einen Depression gut schieben und sammeln mikroskopischen Aufnahmen mit einem Durchlichtmikroskop (Abbildung 2). Sammeln Sie Bilder von 5 Sichtfelder für jedes Experiment an jedem gewünschten Zeitpunkt. </li></ol> 5. Datenanalyse <ol><li> Mit ImageJ oder vergleichbare Bildanalyse-Software, öffnen Sie eine der fünf Bilddatensätze mit dem Import / Bildsequenz ... aus dem Pulldown-Menü Datei. Diese werden als Bildstapels geöffnet werden. </li><li> Im Dialogfeld Bild ... / Eigenschaften, ändern Sie die iMagier Einheiten auf Pixel und setzen Pixel Breite und Höhe bis 1,0 Pixel. </li><li> Konvertieren Sie die Bilder in dual mit dem Anpassen / Schwellenwert ...-Befehl in der Bild Pulldown-Menü. Stellen Sie den Schwellenwert auf Pixel, die Perlen oder Perlen Aggregate beitragen, umfassen, aber das auszuschließen Hintergrund und kleinen Partikeln. Gelten für alle Bilder im Stapel. </li><li> Im Dialogfeld Set Maße, überprüfen Sie die Umgebung und Stack Position Boxen. </li><li> Analyze Particles ... in der Analyse Pulldown-Menü. Dadurch wird eine Liste der identifizierten Partikel, einschließlich ihrer Größe (Fläche) und das Bild in der sie erkannt zu generieren. </li><li> Wiederholen Sie diesen Vorgang für die Experimente und Versuchsbedingung. </li></ol>

<ol>
	<li>Chen, X., Gumbiner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraxial+protocadherin+mediates+cell+sorting+and+tissue+morphogenesis+by+regulating+C-cadherin+adhesion+activity.">Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity.</a> J. Cell Biol. 174 (2), 301-313 (2006).</li><li>Grumet, M., Friedlander, D. R., Edelman, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+the+binding+of+Ng-CAM+to+laminin.">Evidence for the binding of Ng-CAM to laminin.</a> Cell Adhes. Commun. 1 (2), 177-190 (1993).</li><li>Hatta, K., Nose, A., Nagafuchi, A., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+cDNA+encoding+a+neural+calcium-dependent+cell+adhesion+molecule:+its+identity+in+the+cadherin+gene+family.">Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family.</a> J. Cell Biol. 106 (3), 873-881 (1988).</li><li>Hirano, S., Nose, A., Hatta, K., Kawakami, A., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+cell-cell+adhesion+molecules+(cadherins):+subclass+specificities+and+possible+involvement+of+actin+bundles.">Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles.</a> J. Cell Biol. 105 (6 Pt 1), 2501-2510 (1987).</li><li>Nose, A., Nagafuchi, A., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expressed+recombinant+cadherins+mediate+cell+sorting+in+model+systems.">Expressed recombinant cadherins mediate cell sorting in model systems.</a> Cell. 54 (7), 993-1001 (1988).</li><li>Nose, A., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+cadherin+cell+adhesion+molecule:+its+expression+patterns+associated+with+implantation+and+organogenesis+of+mouse+embryos.">A novel cadherin cell adhesion molecule: its expression patterns associated with implantation and organogenesis of mouse embryos.</a> J. Cell Biol. 103 (6 Pt 2), 2649-2658 (1986).</li><li>Schreiner, D., Weiner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+homophilic+interaction+between+gamma-protocadherin+multimers+greatly+expands+the+molecular+diversity+of+cell+adhesion.">Combinatorial homophilic interaction between gamma-protocadherin multimers greatly expands the molecular diversity of cell adhesion.</a> Proc. Natl. Acad. Sci. U. S. A. 107 (33), 14893-14898 (2010).</li><li>Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+correlation+between+cell+adhesive+properties+and+some+cell+surface+proteins.">Functional correlation between cell adhesive properties and some cell surface proteins.</a> J. Cell Biol. 75, 464-474 (1977).</li><li>Sivasankar, S., Brieher, W., Lavrik, N., Gumbiner, B., Leckband, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+molecular+force+measurements+of+multiple+adhesive+interactions+between+cadherin+ectodomains.">Direct molecular force measurements of multiple adhesive interactions between cadherin ectodomains.</a> Proc. Natl. Acad. Sci. U. S. A. 96 (21), 11820-11824 (1999).</li><li>Sivasankar, S., Gumbiner, B., Leckband, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurements+of+multiple+adhesive+alignments+and+unbinding+trajectories+between+cadherin+extracellular+domains.">Direct measurements of multiple adhesive alignments and unbinding trajectories between cadherin extracellular domains.</a> Biophys. J. 80, 1758-1768 (2001).</li><li>Sivasankar, S., Zhang, Y., Nelson, W. J., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+initial+encounter+complex+in+cadherin+adhesion.">Characterizing the initial encounter complex in cadherin adhesion.</a> Structure. 17 (8), 1075-1081 (2009).</li><li>Zhang, Y., Sivasankar, S., Nelson, W. J., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+cadherin+interactions+and+binding+cooperativity+at+the+single-molecule+level.">Resolving cadherin interactions and binding cooperativity at the single-molecule level.</a> Proc. Natl. Acad. Sci. U. S. A. 106 (1), 109-114 (2009).</li><li>Yasuda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-induced+protocadherin+arcadlin+regulates+dendritic+spine+number+by+triggering+N-cadherin+endocytosis+via+TAO2beta+and+p38+MAP+kinases.">Activity-induced protocadherin arcadlin regulates dendritic spine number by triggering N-cadherin endocytosis via TAO2beta and p38 MAP kinases.</a> Neuron. 56 (3), 456-471 (2007).</li><li>Chappuis-Flament, S., Wong, E., Hicks, L. D., Kay, C. M., Gumbiner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+cadherin+extracellular+repeats+mediate+homophilic+binding+and+adhesion.">Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.</a> J. Cell Biol. 154 (1), 231-243 (2001).</li><li>Emond, M. R., Biswas, S., Blevins, C. J., Jontes, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+of+Protocadherin-19+and+N-cadherin+mediates+a+novel+mechanism+of+cell+adhesion.">A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion.</a> J. Cell Biol. 195 (7), 1115-1121 (2011).</li><li>Biswas, S., Emond, M. R., Jontes, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clustered+protocadherins+Pcdhalpha+and+Pcdhgamma+form+a+heteromeric+complex+in+zebrafish.">The clustered protocadherins Pcdhalpha and Pcdhgamma form a heteromeric complex in zebrafish.</a> Neuroscience. 219, 280-289 (2012).</li><li>Blevins, C. J., Emond, M. R., Biswas, S., Jontes, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression,+alternative+splicing,+and+adhesive+properties+of+the+zebrafish+delta1-protocadherins.">Differential expression, alternative splicing, and adhesive properties of the zebrafish delta1-protocadherins.</a> Neuroscience. 199, 523-534 (2011).</li><li>Morishita, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+cadherin-related+neuronal+receptor/protocadherin-alpha+first+extracellular+cadherin+domain+reveals+diversity+across+cadherin+families.">Structure of the cadherin-related neuronal receptor/protocadherin-alpha first extracellular cadherin domain reveals diversity across cadherin families.</a> J. Biol. Chem. 281, 33650-33663 (1074).</li><li>Wojtowicz, W. M., Flanagan, J. J., Millard, S. S., Zipursky, S. L., Clemens, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+Drosophila+Dscam+generates+axon+guidance+receptors+that+exhibit+isoform-specific+homophilic+binding.">Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding.</a> Cell. 118 (5), 619-633 (2004).</li><li>Drees, F., Reilein, A., Nelson, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-adhesion+assays:+fabrication+of+an+E-cadherin+substratum+and+isolation+of+lateral+and+Basal+membrane+patches.">Cell-adhesion assays: fabrication of an E-cadherin substratum and isolation of lateral and Basal membrane patches.</a> Methods Mol. Biol. 294, 303-320 (2005).</li></ol>

The authors declare that they have no competing financial interests.

Zelladhäsion ist ein wesentliches Merkmal der vielzelligen Leben und wird von einem breiten Spektrum von Zelloberflächenproteinen vermittelt. Von diesen sind die detaillierten Klebeeigenschaften nur für einen relativ kleinen Anteil verstanden. Hier haben wir ein einfaches, schnelles Protokoll zur Untersuchung der homophile Haftfähigkeit von sekretierten Ektodomänen in einem geeigneten Epitop-Tag fusioniert beschrieben. Dieser Ansatz hat eine Reihe von wichtigen Vorteilen. Zuerst werden die stabilen Zelllinien nicht...

Zell-Zell-Adhäsion ist wesentlich für die Entwicklung und die Integrität von mehrzelligen Organismen und durch eine Vielfalt von Zelloberflächenmolekülen vermittelt. Viele dieser Adhäsionsmoleküle wurden identifiziert und charakterisiert, obwohl viele noch zu entdecken sind. Verschiedene Methoden werden verwendet, um die Eigenschaften von Zelladhäsionsmolekülen (CAMs), einschließlich Zellsortierung Assays, Zellaggregationsassays 1-8 und biophysikalische Methoden, wie die Rasterkraftmikroskopie und Oberflächenkraftspektroskopie 9-12 untersuchen. Die Komplexität auch in vitro-Systemen unter Verwendung von Zelllinien vereinfacht macht es schwierig, die Hafteigenschaften eines mutmaßlichen CAM bestimmen. Typischerweise wird ein Molekül als ein CAM, wenn die Zellaggregation induziert, wenn sie in einem nicht-klebenden Zelllinie transfiziert. Es ist jedoch klar, dass dies keine direkte Hinweise auf Haftungsaktivität. Beispielsweise erleichtert die Zelloberfläche Lieferung oder Stabilität einesCAM würde auch zu einer erhöhten Zellaggregation 1,13 führen. Darüber hinaus kann eine wahre CAM nicht, Zellaggregation vermitteln, wenn die Zelllinie fehlt anderen Co-Faktoren für Zelloberflächen Lieferung oder Stabilisierung erforderlich. Um diese erschwerenden Faktoren zu vermeiden, mehr direkte Assays eingesetzt werden, die auf der Idee basieren, dass adhäsive Wechselwirkungen sollte eine intrinsische biochemische Eigenschaft der extrazellulären Domäne sein. Während Perlen wurden zunächst zur Ng-CAM 2 zu charakterisieren, haben diese Tests, um Cadherin-vermittelte Adhäsion 12,14,15 untersuchen erweitert. Verwendung Fusion des C-Cadherin-Ektodomäne-Fc-Fusionen zeigten die Gumbiner Labor dass mehrere Cadherin Wiederholungen tragen zu Interaktionen 14 homo. Mit vergleichbaren Wulst Aggregationstests, E-Cadherin und N-Cadherin-Haft wurden ebenfalls gekennzeichnet 12,15, als eine Reihe von Protocadherinen 1,15-18 und Dscam Isoformen von Drosophila haben <em&gt; 19. Hier beschreiben wir einen relativ einfachen und schnellen Test zur Charakterisierung der Klebstoff Aktivität sezerniert Epitop markierte Ektodomäne von mutmaßlichen homo Nocken (Abbildung 1). Wir haben diesen Test verwendet hauptsächlich die Mitglieder der Cadherin-Oberfamilie charakterisieren.

<table><tbody><tr><td>pFc-N1 plasmid</td><td>Addgene</td><td>&lt;span style=&quot;color:red;&quot;&gt;&lt;strong&gt;To be submitted&lt;/strong&gt;&lt;/span&gt;</td><td></td></tr><tr><td>HEK293 cells</td><td>American Type Culture Collection</td><td>CRL-1573</td><td></td></tr><tr><td>DMEM</td><td>Mediatech - Corning</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F2442</td><td></td></tr><tr><td>100x Pen-Strep (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668030</td><td></td></tr><tr><td>Dynabeads &amp;ndash; Protein G</td><td>Life Technologies</td><td>10003D</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3294</td><td></td></tr><tr><td>Depression slides</td><td>Electron Microscopy Sciences</td><td>71878-06</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane</td><td>Millipore</td><td>UFC901024</td><td></td></tr><tr><td>0.45 mm syringe filter</td><td>Sarstedt</td><td>83.1826</td><td></td></tr><tr><td>Dynamag-2 Magnet</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>Fiji (ImageJ) Image Analysis Software</td><td>&lt;a href=&quot;http://fiji.sc/Fiji&quot;&gt;http://fiji.sc/Fiji&lt;/a&gt;</td><td></td><td></td></tr><tr><td colspan="4">Table of Buffers/Solutions</td></tr><tr><td>Growth Media</td><td>DMEM + 10% FBS + Pen-Strep</td><td>Filter sterilize and store at 4 &amp;deg;C.</td><td></td></tr><tr><td>Growth Media &amp;ndash;FBS</td><td>DMEM + Pen-Strep</td><td>Filter sterilize and store at 4 &amp;deg;C.</td><td></td></tr><tr><td>Binding Buffer</td><td>50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2% BSA</td><td>Vortex until dissolved, keep on ice.</td><td></td></tr></tbody></table>

bead aggregation assays for the characterization of putative cell adhesion molecules

Zell-Zell-Adhäsion ist von grundlegender Bedeutung für Vielzeller und wird durch eine Vielfalt von Zelloberflächenproteinen vermittelt. Jedoch sind die adhäsiven Wechselwirkungen vieler dieser Proteine ​​schlecht verstanden. Hier präsentieren wir Ihnen eine einfache, schnelle Methode zur Charakterisierung der Hafteigenschaften von mutmaßlichen homophile Zell-Adhäsionsmoleküle. Kultivierten HEK293-Zellen mit Plasmid-DNA, kodierend eine sekretierte, Epitop-tagged Ektodomäne eines Zelloberflächenprotein transfiziert. Unter Verwendung von für die Epitop-Markierung funktionalisierten Kügelchen wird das lösliche, sekretierte Fusionsprotein aus dem Kulturmedium festgehalten. Die beschichteten Perlen können dann direkt in Perlenaggregationstests oder fluoreszierende Perle Sortier Assays für homophile Adhäsion zu prüfen. Falls gewünscht, kann die Mutagenese dann verwendet, um die spezifische Aminosäuren oder Domänen für die Haftung erforderlichen aufzuklären. Dieser Test erfordert nur geringe Mengen an exprimiertem Protein, nicht die Herstellung von stabilen Zelllinien erfordern, und kann acco werdeninnerhalb von 4 Tagen mplished.

<html> Ein Beispiel ist in Abbildung 2, die Kalzium-abhängige Aggregation zeigt Wulst von der Ektodomäne von N-Cadherin-Fc (NcadEC-Fc) fusioniert dargestellt. In Abwesenheit von Calcium, Perlen weisen eine geringe oder keine Tendenz zu aggregieren, und es gibt keine Zunahme der Gesamtgröße mit der Zeit (1A, C). In Anwesenheit von Calcium, Perlen mit NcadEC-Fc-Show robust Aggregation beschichtet, mit Aggregatgröße über die Zeit (Abbildung 1B, C) ​​zu. Dieses Ex...

Watch this Scientific Journal Video about Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules at JoVE.com

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Wulst Aggregationsassays zur Charakterisierung von putativen Zelladhäsionsmoleküle

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

bead-aggregation-assays-for-characterization-putative-cell-adhesion

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Cell-Cell Interactions

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10.5K Aufrufe.  Ohio State University. Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Serie: Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ability to accurately classify and quantify cell extensions and measure the effect on a cell&#39;s adhesive capacity is critical to determining how cell-signaling events impact cancer cell behavior and aggressiveness. Here, we describe the in vitro design and use of a cell extension quantification method in conjunction with an adhesion capacity assay in an established in vitro model for adrenocortical carcinoma (ACC). Specifically, we test the effects of DKK3, a putative tumor suppressor and a pro-differentiation factor, on the membrane extension phenotype of the ACC cell line, SW-13. We propose these assays to provide relatively simple, reliable, and easily interpretable metrics to measures these characteristics under various experimental conditions.

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

Charakterisierung der Zellmembran Erweiterungen und studieren ihre Rolle im Krebs Zelle Adhäsion Dynamik

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

Forschung

Krebsforschung

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Adhesion Frequency-Assay für In Situ Kinetics Analyse der Cross-Junktionaler Molecular Interactions an der Zell-Zell-Schnittstelle

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Biotechnik

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Biomimetische Materialien zu den Bakterien-Wirt-Interaktionen Charakterisieren

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Immunologie und Infektion

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Test der Haftung unter Scherspannung für das Studium der T-Lymphozyten-Adhäsions-Molekül-Wechselwirkungen

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Dynamische Adhäsion Assay für die funktionelle Analyse von Anti-Adhäsion Therapien bei chronisch entzündlichen Darmerkrankungen

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

Messung transzellulärer Wechselwirkungen durch Proteinaggregation in einem heterologen Zellsystem

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

Neurowissenschaften

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

Die Quantifizierung der Endothelial Cell Haftfähigkeit<em&gt; In-vitro-</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Biologie

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Analyse Zelloberflächenhaftung Remodeling in Reaktion auf mechanische Spannung Mit Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

Quantifizierung von Zell-Substrat-Adhäsionsflächen und Zellformverteilungen in MCF7-Zell-Monolayern

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Wulst Aggregationsassays zur Charakterisierung von putativen Zelladhäsionsmoleküle

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