Ringraziamo il professor Dontscho Kerjaschki, Corina Mayrhofer e Sigurd Krieger presso l&#39;Istituto di Patologia Clinica, Università di Vienna, in Austria per la collaborazione.

Coltura cellulare <ol><li> Crescere le cellule ovariche di criceto cinese (CHO-K1) utilizzando le procedure standard di coltura cellulare in F-12 di media Ham con GlutaMAX ho contenente il 10% di siero fetale bovino, 50 U / ml di penicillina, e 50 mg / ml di streptomicina solfato (Invitrogen). </li><li> Scambio terreno di coltura di siero di supporti liberi. Etichetta proteine ​​di superficie della cellula erano in diversi momenti con CyDye DIGE Fluor Cy3 o Cy5 coloranti minima (vedi cellulare Etichettatura sezione di superficie, sotto). </li><li> Un numero uguale di cellule piscina da ogni punto di tempo e l&#39;etichetta con CyDye DIGE Fluor Cy2. Utilizzare questi come standard interno per ogni 2-D gel. </li><li> La maggior parte degli esperimenti può essere effettuata con cellule CHO-K1, ma fibroblasti embrione di topo (L1 3T3) e ascite linfoma di topo linfoblasti (EL4) può anche essere usato (dati non riportati). </li><li> Crescono i due tipi di cellule quest&#39;ultimo in DMEM media con GlutaMAX II, ma, in caso contrario, l&#39;uso identiche condizioni utilizzate per la cellule CHO-K1. </li></ol> Superficie cellulare etichettatura <ol><li> Con attenzione staccare cellule aderenti non-enzimaticamente, il conteggio e la divisione in aliquote di 5-10 x 106 cellule. Per le cellule crescono in sospensione, omettere il passaggio distacco. </li><li> Centrifugare la sospensione cellulare a circa 800 xg per 5 minuti. Rimuovere il surnatante contenente il mezzo. </li><li> Lavare il pellet da resuspendion in soluzione salina bilanciata 1 ml ghiacciata di Hank (HBSS) pH 8.5. Centrifugato a 800 xg a 4 ° C per 2 minuti. </li><li> Rimuovere il supernatante e risospendere il pellet cellulare in 200 microlitri di buffer etichettatura ghiacciata (HBSS pH 8,5, 1 M urea). </li><li> Etichetta le cellule intatte con 600 pmol CyDye DIGE coloranti Fluor minimo per 20 minuti in ghiaccio al buio. </li><li> Placare la reazione aggiungendo 20 l 10 mM lisina. Incubare per 10 minuti. </li><li> Lavare la superficie marcato le cellule due volte da risospensione in 500 microlitri HBSS pH 7,4, seguita da centrifugazione a 800 xg a 4 ° C per 2 minuti. </li></ol> Lisi cellulare e frazionamento <ol><li> Lisare la superficie marcata con cellule in 150 microlitri di buffer di lisi freddo (7 M urea, 2 M tiourea, 4% CHAPS, 30 mM Tris, 5 mM acetato di magnesio pH 8,5). Lasciare in ghiaccio per almeno 1 h con vortex occasionali. </li><li> Centrifugare il lisato a 10 000 xga 4 ° C per 5 minuti. Trasferire il surnatante in una nuova provetta. Questo esempio è il non-frazionata campione che contiene tutte le proteine ​​cellulari. </li><li> In parallelo, lavare pellet cellulari, come sopra, poi frazionare (utilizzando un kit di frazionamento membrana, Pierce) in frazioni di membrana e citosolici prima di 2-D elettroforesi su gel. La frazione membrana interna e contiene proteine ​​di membrana delle cellule di superficie. </li><li> Per confronto, seguire la procedura standard di Ettan DIGE 3 e lisare, etichetta, e, infine, frazionare le cellule. Determinare la concentrazione di proteine ​​nei campioni con il 2 - D Quant Kit (GE Healthcare). </li></ol> 2-D elettroforesi <ol><li> Reidratare gel DryStrip Immobiline, pH 3-11NL (24 cm) con vassoio Immobiline DryStrip Reswelling, 24 cm in 450 ul di soluzione di reidratazione DeStreak (0,5% IPG buffer) durante la notte. </li><li> Applicare il CyDye marcato campioni (corrispondenti a 50 mg di proteine ​​totali) per Immobiline gel DryStrip caricando coppa anodica nel collettore ed eseguire focalizzazione isoelettrica (IEF) utilizzando Ettan IPGphor II sistema IEF secondo le istruzioni. </li><li> Dopo IEF, equilibrare le strisce in due fasi e posto sulla cima di grandi dimensioni (26 x 20 cm) 12,5% gel di poliacrilammide (SDS-PAGE). Sovrapposizione con il 0,5% agarosio (in esecuzione tampone contenente blu di bromofenolo). Run 2-D elettroforesi usando Ettan DALTtwelve grande sistema verticale a 5 W / gel per 30 minuti, e poi a 15 W / gel fino a quando il fronte del colorante raggiunge il fondo del gel. </li></ol> Imaging e analisi dei dati <ol><li> Dopo aver completato 2-D elettroforesi, la scansione del gel per Cy2, Cy3 o Cy5 utilizzando un tifone 9410 Imager Variable Mode. </li><li> Confrontare le mappe posto da frazioni di membrana, frazioni citosoliche, e non frazionato campioni utilizzando DeCyder 2-D Software differenziale Analisi 4. </li></ol> Post-colorazione <ol><li> Dopo l&#39;imaging, l&#39;argento macchia i gel secondo procedura standard 5. </li></ol> Identificazione delle proteine <ol><li> Coltivare, raccogliere, lavare e lisare quantità preparativa (circa 1 mg di proteine ​​totali da 10 x 106 cellule CHO) di cellule, come descritto in precedenza per un non-frazionata campione. Utilizzare un Cy5 campione superficie cellulare etichettati (vedi sopra) come una punta, e applicare insieme agli importi senza etichetta preparativo di proteine. </li><li> Eseguire l&#39;elettroforesi 2-D come sopra descritto, ma questa volta, strofinavano 600 mg di cellule senza etichetta lisato insieme con 50 mg superficie delle cellule etichettate picco con l&#39;applicazione anodica tazza. Usare i marcatori di riferimento per consentire la raccolta corretta posizione, e il luogo esseretra le lastre di vetro prima del getto gel seguendo le raccomandazioni 3. </li><li> Scansione il gel in Cy5 canale per ottenere la superficie Cy5 mappa cellula posto, seguita dalla colorazione delle proteine ​​totali con profonda proteine ​​totali macchia viola 3. </li><li> Corrispondenza insieme le due mappe spot utilizzando la DeCyder 2-D di software e creare un elenco di tutti i punti corrispondenti ai 5 proteine ​​cellulari Cy etichettati superficie. Scegli le proteine ​​della superficie cellulare utilizzando il punto stazione Ettan manipolazione, estratto le spine gel, e trypsinate utilizzando il digestore Ettan. Identificare con MALDI-TOF spettrometria di massa. </li></ol> Tabella 1. Un esperimento Ettan DIGE è stata effettuata utilizzando campioni di siero di cellule impoverito etichettati secondo la superficie cellulare protocollo di etichettatura delle proteine ​​in figura 1. <table style="width: 517px; height: 139px;" border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="text-align:center; font-weight:bold"> Campione </td><td style="text-align:center; font-weight:bold"> Tempo di esaurimento siero </td><td style="text-align:center; font-weight:bold"> Etichettato con CyDye </td><td style="text-align:center; font-weight:bold"> Gel numero </td></tr><tr><td style="text-align:center;"> 1 </td><td style="text-align:center;"> - </td><td style="text-align:center;"> Cy 3, Cy 2 </td><td style="text-align:center;"> 1 </td></tr><tr><td style="text-align:center;"> 2 </td><td style="text-align:center;"> 30 minuti </td><td style="text-align:center;"> Cy 5, Cy 2 </td><td style="text-align:center;"> 1 </td></tr><tr><td style="text-align:center;"> 3 </td><td style="text-align:center;"> 2 ore </td><td style="text-align:center;"> Cy 3, Cy 2 </td><td style="text-align:center;"> 2 </td></tr><tr><td style="text-align:center;"> 4 </td><td style="text-align:center;"> 4 ore </td><td style="text-align:center;"> Cy 5, Cy 2 </td><td style="text-align:center;"> 2 </td></tr><tr><td style="text-align:center;"> 5 </td><td style="text-align:center;"> 16 ore </td><td style="text-align:center;"> Cy 5, Cy 2 </td><td style="text-align:center;"> 3 </td></tr></tbody></table>

<ol>
	<li>Jang, J. H., Hanash, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+the+cell+surface+proteome.">Profiling of the cell surface proteome.</a> Proteomics. 3, 1947-1954 (2003).</li><li>Shin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+profiling+of+the+cell+surface+proteome+of+cancer+cells+uncovers+an+abundance+of+proteins+with+chaperone+function.">Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function.</a> J Biol Chem. 9, 7607-7616 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ettan+DIGE+System+User+Manual.">Ettan DIGE System User Manual.</a> GE Healthcare user manual 18-1164-40. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeCyder+2-D+Differential+Analysis+Software+v+6.0.+GE+Healthcare+data+file+11-0011-98+Edition+AA.">DeCyder 2-D Differential Analysis Software v 6.0. GE Healthcare data file 11-0011-98 Edition AA.</a> , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-D+Electrophoresis+using+immobilized+pH+gradients,+principles+and+methods.">2-D Electrophoresis using immobilized pH gradients, principles and methods.</a> GE Healthcare handbook 80-6429-60 Edition AB. , (2002).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CyDye+DIGE+fluors+and+labeling+kits.+GE+Healthcare+data+file+18-1164-84+Edition+AB.">CyDye DIGE fluors and labeling kits. GE Healthcare data file 18-1164-84 Edition AB.</a> , (2003).</li><li>Mayrhofer, C., Krieger, S., Allmaier, G., Kerjaschki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIGE+compatible+labeling+of+surface+proteins+on+vital+cells+in+vitro+and+in+vivo.">DIGE compatible labeling of surface proteins on vital cells in vitro and in vivo.</a> Proteomics. 2, 579-5785 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+cell-surface+proteins+using+CyDye+DIGE+Fluor+minimal+dyes.+GE+Healthcare+Application+Note+11-0033-92+Edition+AB.">Selective labeling of cell-surface proteins using CyDye DIGE Fluor minimal dyes. GE Healthcare Application Note 11-0033-92 Edition AB.</a> , (2005).</li></ol>

Concentrazione di proteine Una panoramica dei due flussi di lavoro di etichettatura è illustrato nella figura 1. Dal momento che le cellule sono ancora intatte quando etichettati secondo la superficie cellulare protocollo proteina etichettatura, solo le proteine ​​della superficie cellulare sono esposti al colorante. Nello standard Ettan DIGE protocollo, le cellule sono lisate prima di etichettatura e di proteine ​​all&#39;interno e all&#39;esterno della cellula sono etichettati (Fig....

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CyDye DIGE Kit, 2 nmol</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>28-9345-30</td>
<td>&nbsp;</td>
<tr>
<td>2-D Quant Kit</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>80-6484-51</td>
<td>&nbsp;</td>
<tr>
<td>IPG Buffer pH 3-11NL</td>
<td>Reagent</td>
<td>GE Heallthcare</td>
<td>17-6004-40</td>
<td>&nbsp;</td>
<tr>
<td>Immobiline DryStrip pH 3-11NL, 24 cm</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-6003-77</td>
<td>&nbsp;</td>
<tr>
<td>IPGbox</td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>28-9334-65</td>
<td>&nbsp;</td>
<tr>
<td>IPGbox kit</td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>28-9334-92</td>
<td>&nbsp;</td>
<tr>
<td>DeStreak Rehydration solution</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-6003-19</td>
<td>&nbsp;</td>
<tr>
<td>Immobiline DryStrip Cover Fluid</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1335-01</td>
<td>&nbsp;</td>
<tr>
<td>Ettan IPGphor 3 IEF Unit</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>11-0033-64</td>
<td>&nbsp;</td>
<tr>
<td>Ettan IPGphor Manifold</td>
<td>Instrument component</td>
<td>GE Healthcare</td>
<td>80-6498-38</td>
<td>&nbsp;</td>
<tr>
<td>Ettan DALTtwelve Gel Caster </td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>80-6467-22</td>
<td>&nbsp;</td>
<tr>
<td>Ettan DALTtwelve Separation Unit and Power Supply/Control Unit</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>80-6466-46</td>
<td>230 V unit: 80-6466-27</td>
</tr>
<tr>
<td>Typhoon 9410 Variable Mode Imager</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>63-0055-81</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Spot Picker</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1145-28</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Digester</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-68</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Spotter</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-67</td>
<td>&nbsp;</td>
<tr>
<td>Ettan MALDI-TOF</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-33</td>
<td>&nbsp;</td>
<tr>
<td>DeCyder 2-D Differential Analysis Software</td>
<td>Other</td>
<td>GE Healthcare</td>
<td>28-4012-01</td>
<td>&nbsp;</td>
<tr>
<td>ImageQuant Analysis Software</td>
<td>Other</td>
<td>GE Healthcare</td>
<td>28-9236-62</td>
<td>&nbsp;</td>
<tr>
<td>Urea</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1319-01</td>
<td>&nbsp;</td>
<tr>
<td>CHAPS</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1314-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Dithiothreitol (DTT)</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1318-01</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol Blue</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1329-01</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1321-01</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Dodecylsulfate (SDS)</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1313-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Glycerol 87%</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1325-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne ReadySol IEF 40% T, 3% C</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1310-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne TEMED</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1312-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Ammonium Persulfate</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1311-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Glycine </td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1323-01</td>
<td>&nbsp;</td>
<tr>
<td>2-D Sample Prep for membrane proteins</td>
<td>Reagent</td>
<td>Pierce</td>
<td>89864</td>
<td>&nbsp;</td>
<tr>
<td>Streptomycin sulphate</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>		</tbody>
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selective labelling of cell-surface proteins using cydye dige fluor minimal dyes

Proteine ​​di superficie sono fondamentali per la capacità della cellula di reagire al suo ambiente e di interagire con le cellule vicine. Essi sono noti per essere induttori di quasi tutti i segnali intracellulari. Inoltre, essi svolgono un ruolo importante nell&#39;adattamento ambientale e trattamento farmacologico, e spesso sono coinvolti nella patogenesi della malattia e patologia (1). Interazioni proteina-proteina sono intrinseche vie di segnalazione, e per ottenere un quadro più chiaro in questi complessi processi biologici, metodi sensibili e affidabili sono necessari per lo studio delle proteine ​​di superficie delle cellule. Bidimensionale (2-D) elettroforesi è ampiamente utilizzato per il rilevamento dei biomarcatori e altri obiettivi in ​​campioni di proteine ​​complesse per studiare i cambiamenti differenziale. Proteine ​​di superficie cellulare, in parte a causa della loro abbondanza basso (1 2% di proteine ​​cellulari), sono difficili da individuare in un 2-D gel senza frazionamento o qualche altro tipo di arricchimento. Sono anche spesso poco rappresentati in 2-D gel a causa della loro natura idrofobica e ad alto peso molecolare (2). In questo studio, presentiamo un nuovo protocollo per le cellule intatte mediante CyDye DIGE coloranti Fluor minimi specifici per l&#39;etichettatura e la rilevazione di questo importante gruppo di proteine. I risultati hanno mostrato specifici dell&#39;etichettatura di un gran numero di proteine ​​di superficie delle cellule con l&#39;etichettatura minimo di proteine ​​intracellulari. Questo protocollo è rapido, semplice da usare, e tutti CyDye tre DIGE coloranti Fluor minima (Cy 2, 3 e Cy Cy 5) può essere usato per etichettare superficie cellulare delle proteine. Queste caratteristiche consentono di multiplexing utilizzando il 2-D elettroforesi su gel a fluorescenza Differenza (2-D DIGE) con tecnologia DIGE Ettan e l&#39;analisi dei cambiamenti dell&#39;espressione proteica con DeCyder 2-D Software differenziale di analisi. Il livello di superficie cellulare delle proteine ​​è stato seguito durante il digiuno nel siero di cellule CHO per varie lunghezze di tempo (vedi tabella 1). Piccoli cambiamenti in abbondanza sono stati rilevati con elevata precisione, ed i risultati sono supportati da definire metodi statistici.

Watch this Scientific Journal Video about Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes at JoVE.com

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Un metodo semplice e specifico è stato dimostrato per la marcatura fluorescente e la rilevazione maggiore di proteine ​​di superficie cellulare, senza un passo di frazionamento. Abbondanza differenziale di proteine ​​della superficie cellulare è stato analizzato mediante elettroforesi bidimensionale (2-D) e Ettan ™ tecnologia DIGE.

Etichettatura selettiva di proteine ​​della superficie cellulare utilizzando CyDye DIGE Coloranti Fluor Minimal

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

selective-labelling-cell-surface-proteins-using-cydye-dige-fluor

Research

JoVE Journal

Biology

15.1K Views.  GE Healthcare Bio-Sciences AB. Un metodo semplice e specifico è stato dimostrato per la marcatura fluorescente e la rilevazione maggiore di proteine ​​di superficie cellulare, senza un passo di frazionamento. Abbondanza differenziale di proteine ​​della superficie cellulare è stato analizzato mediante elettroforesi bidimensionale (2-D) e Ettan ™ tecnologia DIGE.

Video: Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

La registrazione ottica di attività elettrica in Guinea-maiale Reti enterici utilizzando tensione sensibile Coloranti

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Ricerca

Biologia

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

Avidità-based Screening Interaction extracellulare (AVEXIS) per il rilevamento scalabile di bassa affinità extracellulare del recettore-ligando interazioni

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Cattura selettiva di 5 hydroxymethylcytosine dal DNA genomico

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Glicopeptide Capture per Proteomica cellulari di superficie

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Misurazione della extracellulare Ion Flussi Utilizzando il ionoselettivo autoreferenziale microelettrodo Tecnica

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

Sintesi della lunghezza d&#39;onda-shifting DNA sonde di ibridazione utilizzando Fotostabile Cyanine Coloranti

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Determinazione del relativo cellulari di superficie e di espressione totale di canali ionici ricombinante in citometria a flusso

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

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 ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

Applicazione di coloranti fluorescenti selettivi a membrana e a parete cellulare per l'imaging a celle vive di Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Etichettatura selettiva di proteine ​​della superficie cellulare utilizzando CyDye DIGE Coloranti Fluor Minimal

Etichettatura selettiva di proteine della superficie cellulare utilizzando CyDye DIGE Coloranti Fluor Minimal