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 border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:212px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:216px;">Company</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">pFc-N1 plasmid</td>
			<td style="width:203px;">To be submitted</td>
			<td style="width:216px;">Addgene</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">HEK293 cells</td>
			<td style="width:203px;">CRL-1573</td>
			<td style="width:216px;">American Type Culture Collection</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">DMEM</td>
			<td style="width:203px;">10-013-CV</td>
			<td style="width:216px;">Mediatech - Corning</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">F2442</td>
			<td style="width:216px;">Sigma</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">100X Pen-Strep (10,000 U/ml)</td>
			<td style="width:203px;">15140-122</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">0.05% Trypsin-EDTA</td>
			<td style="width:203px;">25300054</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Lipofectamine 2000</td>
			<td style="width:203px;">11668030</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Dynabeads &#8211; Protein G</td>
			<td style="width:203px;">10003D</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Bovine Serum Albumin</td>
			<td style="width:203px;">A3294</td>
			<td style="width:216px;">Sigma</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Depression slides</td>
			<td style="width:203px;">71878-06</td>
			<td style="width:216px;">Electron Microscopy Sciences</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:212px;">Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane</td>
			<td rowspan="2" style="width:203px;">UFC901024</td>
			<td rowspan="2" style="width:216px;">Millipore</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">0.45mm syringe filter</td>
			<td style="width:203px;">83.1826</td>
			<td style="width:216px;">Sarstedt</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Dynamag-2 Magnet</td>
			<td>12321D</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:212px;">Fiji (ImageJ) Image Analysis Software</td>
			<td style="width:203px;"></td>
			<td style="width:216px;">http://fiji.sc/Fiji</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Table of Buffers/Solutions</td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Growth Media</td>
			<td style="width:203px;">DMEM + 10% FBS + Pen-Strep</td>
			<td style="width:216px;">Filter sterilize and store at 4&#176;C.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Growth Media &#8211; FBS</td>
			<td style="width:203px;">DMEM + Pen-Strep</td>
			<td style="width:216px;">Filter sterilize and store at 4&#176;C.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:212px;">Binding Buffer</td>
			<td style="width:203px;">50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2% BSA</td>
			<td style="width:216px;">Vortex until dissolved, keep on ice.</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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Cell-Cell Interactions

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10.4K 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

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Sintesi, assemblaggio e caratterizzazione di film monostrato Protetta delle nanoparticelle d&#39;oro per Elettrochimica proteine ​​monostrato

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

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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 ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Utilizzando impedenza Cell-substrato e imaging cellulare dal vivo per misurare i cambiamenti in tempo reale in Cellular Adesione e De-adesione indotta da Matrix Modifica

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Ambiente controllato Microfluidic per Dynamic Indagine Globulo rosso Aggregazione

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Wet Chimica e Peptide immobilizzazione su politetrafluoroetilene per Miglioramento di adesione cellulare

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D Stem magnetica cellulare Aggregazione e bioreattore maturazione per la cartilagine Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

Sviluppo di dispositivi microfluidici per studiare la capacità di allungamento di punta-coltivazione di cellule vegetali in spazi estremamente ridotti

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Rilevamento dell'endotossina in Nano-formulazioni utilizzando dosaggi di Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Una piattaforma microfluidica multistrato per la conduzione di un'espressione genica prolungata senza cellule

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 &#181;m in length with inner hydrophobic hollow channels and outer hydrophilic surfaces. JBNTs are positively charged and FNs are negatively charged. Therefore, when injected into a neutral aqueous solution, they are bonded together via noncovalent bonding to form the NM bundles. The self-assembly process is completed within a few seconds without any chemical initiators, heat source, or UV light. When the pH of the NM solution is lower than the isoelectric point of FNs (pI 5.5-6.0), the NM bundles will self-release due to the presence of positively charged FN.
NM is known to mimic the extracellular matrix (ECM) morphologically and hence, can be used as an injectable scaffold, which provides an excellent platform to enhance hMSC adhesion. Cell density analysis and fluorescence imaging experiments indicated that the NMs significantly increased the anchorage of hMSCs compared to the negative control.

The goal of this protocol is to show the assembly of a biomimetic nanomatrix (NM) with Janus base nanotubes (JBNTs) and fibronectin (FN). When co-cultured with human mesenchymal stem cells (hMSCs), the NMs exhibit excellent bioactivity in encouraging hMSCs adhesion.

Fabbricazione di una nanoma matrice biomimetica con nanotubi di base Janus e fibronectina per l'adesione delle cellule staminali

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...