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

1 Cella di preparazione (Giorno -2 a 0) <ol><li> Split cellule HEK293 1: 5 con 0,05% di soluzione di tripsina-EDTA e incubare in crescita media a 37 ° C con il 5% di CO 2 fino al 60 - 80% confluenti (2 - 3 giorni). Per ogni condizione, le cellule di coltura in 2 x 100 piatti mm. </li></ol> 2. cellulare trasfezione (giorno 1) <ol><li> Trasfezione le cellule HEK293 con plasmide codificante Fc-fusion che usano un reagente di trasfezione quali Lipofectamine. Metodi alternativi che si traducono in efficienza di transfezione comparabili possono anche essere utilizzati. </li><li> Ritorna cellule trasfettate per incubatore per 24 ore. </li></ol> 3. cellulare Propagazione (Day 2) <ol><li> Preriscaldare mezzi di crescita senza siero fetale bovino (-FBS) a 37 ° C. </li><li> Sciacquare piatti 2x 10 ml Crescita media -FBS, e restituiscono i piatti per la 37 ° C incubatore per 1 ora. </li><li> Sciacquare la culture piatti ancora una volta con 10 ml Crescita media -FBS, per un totale di 3 lavaggi. </li><li> Incubate le cellule HEK293 trasfettate a 37 ° C per altre 48 ore senza FBS prima di raccogliere i media. </li></ol> 4. Perlina Aggregation (Giorno 4) <ol><li> Per raccogliere i supporti dalle piastre di coltura, trasferire file multimediali da ogni coppia di piatti da 50 ml provette coniche e spin a 500 xg per 5 minuti per far sedimentare i detriti cellulari. </li><li> Filtranti da 50 ml tubo conico in un filtro centrifugo con una siringa da 30 ml e 0,45 micron filtro a siringa. </li><li> Spin filtri centrifughi a 4.000 xg e 4 ° C fino al volume di terreno di coltura concentrato è di circa 500 microlitri (circa 15 minuti). Ripetere fino a quando è stato aggiunto tutti i media cultura e concentrato. </li><li> Aggiungere 1,5 ml di proteina G sfere magnetiche a 1 ml di freddo ghiaccio Binding Buffer in una provetta da 1,5 ml microcentrifuga per ogni campione, posto su un magnete e rimuovere il tampone. Aggiungere immediatamente il terreno di coltura concentrato alla proteina G sfere magnetiche. </li><li> Ruotare i tubi a 4 ° C per 2 ore. </li><li> Pltubi asso su un magnete e rimuovere i supporti. Lavare rapidamente perle due volte con il freddo ghiaccio 1 ml di Binding Buffer, e poi risospendere perline in 300 microlitri Binding Buffer. </li><li> Split perline risospeso in 2 tubi, 150 ml in ciascun tubo, e quindi aggiungere 1,5 ml di mM CaCl 2 o 200 mM EDTA 200 per il &quot;calcio&quot; e condizioni &quot;non calcio&quot;, rispettivamente. </li><li> Trasferire 100 microlitri di ciascuna condizione di una depressione ben scorrere raccogli micrografie utilizzando un microscopio a luce trasmessa (Figura 2). Raccogliere immagini da 5 campi di visualizzazione per ogni esperimento in ogni punto desiderato. </li></ol> Analisi dei dati 5. <ol><li> Utilizzando ImageJ, o software di analisi dell&#39;immagine paragonabile, aprire uno dei cinque set di dati di immagine utilizzando l&#39;Importazione / Sequenza d&#39;immagini ... dal menu a tendina File. Questi saranno aperti come una serie di immagini. </li><li> Nella finestra di dialogo ... Immagine / Proprietà modificare l&#39;iunità mage per pixel e impostare Pixel Larghezza e Altezza pixel a 1.0. </li><li> Convertire le immagini in binario usando il / Soglia Regola ... comando nel menu a tendina immagine. Impostare la soglia per includere i pixel che contribuiscono a perline o aggregati tallone, ma che escludono background e piccole particelle. Applica a tutte le immagini nello stack. </li><li> Nella finestra di dialogo Imposta misurazioni, selezionare le caselle di area e Stack di posizione. </li><li> Eseguire Analizza particelle ... nel Analizza menu a tendina. Questo genererà un elenco di particelle identificate, compresa la loro dimensione (area) e l&#39;immagine in cui sono stati identificati. </li><li> Ripetere questo processo per il quale sperimentare e condizione sperimentale. </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.

L&#39;adesione cellulare è una caratteristica essenziale della vita pluricellulare ed è mediata da una vasta gamma di proteine ​​di superficie cellulare. Di questi, le proprietà adesive dettagliate sono compresi solo una parte relativamente piccola. Qui, abbiamo descritto un protocollo rapido semplice per indagare la capacità adesiva homophilic di ectodomains secreti fuse a un tag epitopo conveniente. Questo approccio ha una serie di importanti vantaggi. In primo luogo, le linee cellulari stabili non sono necess...

Adesione cellula-cellula è essenziale per lo sviluppo e l&#39;integrità degli organismi multicellulari ed è mediata da una gamma diversificata di molecole di superficie cellulare. Molte di queste molecole di adesione sono stati identificati e caratterizzati, anche se molti rimangono da scoprire. Diversi metodi sono utilizzati per studiare le proprietà delle molecole di adesione cellulare (CAM), tra cui separazione delle cellule saggi, saggi di aggregazione delle cellule 1-8 e metodi biofisici, come la microscopia a forza atomica e spettroscopia di forza di superficie 9-12. La complessità di anche semplificata nei sistemi in vitro su linee cellulari rende difficile determinare le proprietà adesive di una CAM putativo. Tipicamente, una molecola è considerata una CAM se induce aggregazione cellulare quando trasfettati in una linea cellulare non adesivo. Tuttavia, è chiaro che questa non è una prova diretta dell&#39;attività adesivo. Per esempio, facilitando la consegna superficie cellulare o la stabilità di unCAM potrebbe anche tradursi in una maggiore aggregazione delle cellule 1,13. Inoltre, un vero CAM potrebbe non riuscire a mediare aggregazione cella se la linea cellulare manca di altri co-fattori necessari per la consegna superficie cellulare o stabilizzazione. Per evitare questi fattori di complicazione, le analisi più dirette possono essere impiegate che si basano sull&#39;idea che le interazioni adesive dovrebbe essere una proprietà biochimica intrinseca del dominio extracellulare. Mentre perline sono stati inizialmente utilizzati per caratterizzare Ng-CAM 2, questi test sono stati estesi al fine di indagare caderina-mediata adesione 12,14,15. Utilizzando fusione dei C-caderina ectodomain-Fc fusioni, il laboratorio Gumbiner ha mostrato che più ripetizioni caderina contribuiscono a omofiliche interazioni 14. Utilizzando comparabili metodiche di aggregazione tallone, E-caderina e N-caderina adesione sono stati anche caratterizzati 12,15, come avere un numero di protocadherins 1,15-18 e isoforme Dscam di Drosophila <em&gt; 19. Qui descriviamo un test relativamente semplice e rapida per caratterizzare l&#39;attività adesiva di secreti, ectodomains epitopi-tag di camme omofiliche putativi (Figura 1). Abbiamo usato questo test principalmente per caratterizzare i membri della superfamiglia caderina.

<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

Adesione cellula-cellula è fondamentale per la vita pluricellulare ed è mediata da una gamma diversificata di proteine ​​di superficie cellulare. Tuttavia, le interazioni adesive per molte di queste proteine ​​sono poco conosciuti. Qui vi presentiamo un metodo rapido semplice per caratterizzare le proprietà adesive delle molecole di adesione cellulare omofiliche putativi. HEK293 cellule coltivate vengono trasfettate con il plasmide di DNA che codifica un secreto, epitopo-tagged ectodomain di una proteina di superficie cellulare. Utilizzando perline funzionalizzati specifici per il tag epitopo, il, proteina di fusione solubile secreta viene catturato dal mezzo di coltura. Le perle rivestite possono essere utilizzati direttamente nel tallone metodiche di aggregazione o in cordone fluorescente smistamento test per verificare l&#39;adesione homophilic. Se lo si desidera, mutagenesi può quindi essere utilizzato per chiarire gli amminoacidi specifici o domini richiesti per l&#39;adesione. Questo test richiede solo piccole quantità di proteina espressa, non richiede la produzione di linee cellulari stabili, e può essere ACCOmplished in 4 giorni.

<html> Un esperimento di esempio è presentato in figura 2, che mostra tallone aggregazione calcio-dipendente dal ectodomain di N-caderina fuso a Fc (NcadEC-Fc). In assenza di calcio, perline mostrano poca o nessuna tendenza ad aggregarsi e non vi è alcun aumento delle dimensioni aggregata con il tempo (Figura 1A, C). In presenza di calcio, perline rivestite con NcadEC-Fc mostra robusta aggregazione, con granulometria crescente nel tempo (Figura 1B, C). Questo esperimen...

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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.

Perlina di aggregazione saggi per la caratterizzazione di molecole di adesione cellulare putativo

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

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10.5K Views.  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.

Video: 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.

Caratterizzazione delle estensioni della membrana cellulare e studiando i loro ruoli nella dinamica di adesione delle cellule cancro

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

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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.

Adesione saggio di frequenza per In Situ Analisi cinetica delle interazioni molecolari incrociati giunzionale alla cellula-cellula di interfaccia

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

Bioingegneria

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.

I materiali biomimetici per caratterizzare le interazioni batteri-ospite

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 ...

Immunologia e infezioni

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.

Saggio di adesione Sotto Shear stress per lo studio di linfociti T-Adhesion Molecule Interazioni

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.

Analisi di adesione dinamico per l'analisi funzionale delle terapie di anti-adesione nelle malattie intestinali infiammatorie

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.

Misurare le interazioni transcellulari attraverso l'aggregazione proteica in un sistema di cellule eterologi

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

Neuroscienze

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.

La quantificazione di adesività delle cellule endoteliali<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 ...

Biologia

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.

Analizzando cellulare superficie di adesione ritocca in risposta alla tensione meccanica utilizzando biglie magnetiche

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.

Quantificazione dell'area di adesione del substrato cellulare e della distribuzione delle forme cellulari nei monostrati cellulari MCF7

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.

Perlina di aggregazione saggi per la caratterizzazione di molecole di adesione cellulare putativo

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