Il lavoro è il nostro laboratorio è supportato anche da finanziamenti del National Institutes of Health.

Preparazione <ul><li> Coprioggetto devono essere sterilizzati in autoclave. </li><li> Acqua distillata o deionizzata deve essere utilizzato per preparare le soluzioni e per coprioggetto lavaggio. </li><li> AC (40% w / v) e bis-CA (1% w / v) le soluzioni sono sterilizzate di 0,2 micron di filtrazione. Preparare persolfato di ammonio al 10% (APS; acqua 100μg/ml) poco prima dell&#39;uso e filtro sterile. Sostituire la soluzione APS mensile. </li><li> Reagenti chimici come 3-APTMS, cloroformio, glutaradehyde, NHS, e SurfaSil che non possono essere sterilizzati in autoclave sono conservati in una bottiglia assegnato utilizzati esclusivamente per la preparazione di idrogeli. </li><li> Per ottenere risultati ottimali, gli idrogel deve essere utilizzato entro un paio di giorni dopo la notte di incubazione con la proteina appropriato ECM. </li><li> Preparare una soluzione SurfaSil 10% in cloroformio (10 ml di solito è sufficiente per 20 lamelle in alto) in un tubo in polipropilene da 50 ml Falcon prima colata di idrogel. (Il nostro laboratorio siliconizes di solito i coprioggetti alto durante la fase di incubazione glutaraldeide 0,5%). Aggiungere i coprioggetti al tubo Falcon e rock per almeno 10 min. Decantare la soluzione SurfaSil e aria secca il coprioggetto sul Kimwipes nella cappa di sicurezza biologica dove l&#39;idrogel sarà preparato. </li><li> Preparare il calore inattivato soluzione BSA come segue: a 20 mg / ml soluzione di acidi grassi liberi BSA in PBS è incubato in un bagno d&#39;acqua ° 68 C per 30 min. La soluzione è quindi sterile filtrato e conservato a 4 ° C. </li></ul> Procedura <ol><li> Mettere uno strato di Parafilm nella metà inferiore di un 150 mm piastra di Petri. </li><li> Posto fino a 9 da 25 mm coprioggetto sulla parte superiore del Parafilm e coprirli con 1 ml 0,1 M NaOH. Incubare per 3 minuti e poi aspirare con una linea a vuoto. </li><li> Lavorare in una cappa chimica, posto 0,5 ml 3-APTMS su ogni vetrino. Incubare per 3 minuti e poi aspirare il APTMS. Se aspettate troppo a lungo, si formerà una schiuma. </li><li> Sciacquare i coprioggetti una volta con 20 ml di acqua deionizzata nello stesso piatto. Togliere i coprioggetti dal piatto con pinze curve e trasferirle, con i loro trattati sul lato rivolto verso l&#39;alto, ad un nuovo piatto da 150 mm. Lavare i coprioggetti con acqua deionizzata per tre volte, sul bilanciere, per 10 minuti ogni lavaggio. Se non si riesce a rimuovere tutti i APTMS, che reagirà con la glutaraldeide nel passaggio successivo e lasciare un precipitato bianco nuvoloso (Figura 1). </li><li> Scongelare la glutaraldeide (~ 10 minuti prima dell&#39;uso). </li><li> Utilizzando pinze curve, trasferire i coprioggetti di un piatto pulito strati con Parafilm ed aspirare il liquido rimanente. Utilizzare una linea di vuoto o Kimwipe a cancellare l&#39;acqua residua, se necessario. </li><li> Coprire ogni copri completamente con 0,5 ml di glutaraldeide 0,5% in acqua deionizzata sterile e incubare per 30 minuti in una cappa chimica. Aspirare il glutaraldeide. Sciacquare e lavare i coprioggetti come al punto 4. Asciugare il coprioggetto completamente. [Si può fermare qui e lasciare il coprioggetto per diverse settimane in un luogo asciutto]. </li><li> Quando si è pronti per preparare il idrogel, utilizzare pinze curve per trasferire i coprioggetti, fino lato reattivo, ad un foglio di Parafilm che è stato registrato sulla superficie della cappa di sicurezza biologica. Assicurarsi che il coprioggetto sono piatte sulla superficie Parafilm. </li><li> Preparare una soluzione satura NHS in toluene. (Sciogliere una piccola quantità di NHS in toluene sufficiente per l&#39;esperimento particolare. Continuare ad aggiungere po &#39;NHS a poco fino a quando il SSN non si scioglie. La soluzione satura di solito è nuvoloso e rosa.) </li><li> Quindi, preparare l&#39;, acrilamide bis-acrilamide, acqua e APS per raggiungere la percentuale di acrilammide desiderato. Aggiungere i reagenti in provette da microcentrifuga come indicato di seguito per un volume totale di 0.8ml. </li><li> Poi, un aliquota alla volta, aggiungere il SSN e TEMED alla soluzione di 0,8 ml AC, vortice brevemente e versare IMMEDIATAMENTE 3-5 gel con 140 microlitri per coprioggetto all&#39;interno della cappa di sicurezza biologica. Per quanto possibile, tutti i passi da questo punto deve essere eseguita in una cappa di sicurezza biologica. [Nota: lamelle di diverse dimensioni possono essere utilizzati basato sul bisogno. Se da 18 mm coprioggetto sono utilizzati, utilizzare ~ 33 ul di soluzione CA per coprioggetto e una da 18 mm coprioggetto top siliconata.] <table border="1"><tbody><tr><td></td><td colspan="4"> Bis-AC (%) </td></tr><tr><td></td><td> 0,3 (rigido) </td><td> 0,15 </td><td> 0,06 </td><td> 0,03 (soft) </td></tr><tr><td></td><td> microlitri </td><td> microlitri </td><td> microlitri </td><td> microlitri </td></tr><tr><td> Acqua </td><td> 402 </td><td> 522 </td><td> 594 </td><td> 618 </td></tr><tr><td> AC </td><td> 150 </td><td> 150 </td><td> 150 </td><td> 150 </td></tr><tr><td> Bis-AC </td><td> 240 </td><td> 120 </td><td> 48 </td><td> 24 </td></tr><tr><td> APS </td><td> 8 </td><td> 8 </td><td> 8 </td><td> 8 </td></tr><tr><td> TEMED</td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> SSN </td><td> 228 </td><td> 228 </td><td> 228 </td><td> 228 </td></tr></tbody></table></li><li> Rapidamente il luogo siliconato da 25 mm coprioggetto sopra l&#39;gel prima che inizi a polimerizzare. Aggiunta del coprioggetto superiore dovrebbe consentire l&#39;AC per coprire completamente il fondo coprioggetto. Incubare questo &quot;sandwich&quot; a temperatura ambiente fino a quando il CA polimerizza. (Controllare la soluzione residua AC nella provetta per determinare quando la polimerizzazione si è verificato. Solito pochi minuti per i gel rigidi e un po &#39;più morbido per i più sarà sufficiente.). </li><li> Cura ritirare il panino (guanti sterili su!) E sfilare il coprioggetto in alto fino a che sovrasta il gel polimerizzato. È quindi possibile staccare il gel. Se si attende troppo a lungo prima di togliere il copri superiore, il gel si strappare il vetrino è stato rimosso. </li><li> Eliminare il coprioggetto in alto. Posizionare la parte inferiore del gel-coprioggetto (di seguito chiamato idrogel) in 6 pozzetti con 2 ml di PBS / bene. PBS può essere aggiunto prima o dopo il gel-coprioggetto in ciascun pozzetto. Lavare la idrogel con PBS per tre volte, su una sedia a dondolo, a 5 minuti a lavaggio. </li><li> Ripetere i passaggi fino a 11-14 il numero richiesto di idrogel sono stati preparati. </li><li> Coprire ogni idrogel con 2 ml di una soluzione di fibronectina (3 mg / ml in PBS) o proteine ​​ECM altri (si veda Klein et al. 13 per maggiori dettagli.). La proteina ECM diventa un legame covalente con l&#39;idrogel durante l&#39;incubazione overnight a 4 ° C. </li><li> Aspirare la soluzione di ECM e il blocco del NHS non ha reagito con 1mg/ml inattivato al calore di acidi grassi liberi nel siero BSA-multimediale gratuito per almeno 30 minuti a 37 ° C in un incubatore coltura cellulare. Sciacquare il idrogel una volta con PBS sterile o terreno di coltura cellulare. </li><li> Cellule piatto nel mezzo di coltura contenente FBS appropriato. Il numero di cellule seminate sul idrogel deve essere determinato dall&#39;utente in base al grado di diffusione delle cellule e di confluenza richiesto per la sperimentazione. Circa 10 5 cellule di solito è sufficiente per l&#39;analisi western blotting e qPCR. </li><li> Dopo il periodo di incubazione, la proteina cellulare o mRNA può essere estratto. I coprioggetti sono accuratamente rimosse dai pozzi con pinze curve e posto (cella rivolto verso il basso) sulla parte superiore di 100 gocce l di tampone di lisi che sono state intervallate (2-3cm) lungo un foglio di Parafilm sul banco di laboratorio. (Noi uso standard tampone campione SDS e TRIzol, rispettivamente, per preparare i campioni per blotting occidentale e per isolare l&#39;RNA per qPCR.) Incubare le cellule con il tampone di lisi per esattamente un minuto. Togliere i coprioggetti e trasferire il tampone di lisi di una provetta. In alternativa per l&#39;estrazione di RNA solo, l&#39;utente può trasferire ogni idrogel ad un nuovo cambio a 6 pozzetti e aggiungere 1 ml di TRIzol / bene. Incubare per 3 minuti e rimuovere TRIzol soluzione per lo stoccaggio in una provetta. </li></ol> Ulteriori informazioni sulle procedure quali immunofluorescenza, colorazione BrdU, trasfezione, ecc per le cellule seminate su idrogel è descritto in Klein et al. 2007 13. Rappresentante Risultati Lavaggio accurato delle coprioggetto seguenti aggiunta di APTMS è un passo importante nella produzione di &quot;reattivo&quot; coprioggetto. Se uno non riesce a rimuovere il APTMS completamente, reagirà con la glutaraldeide nel passaggio seguente e produrre un precipitato bianco torbido come si vede nella Figura 1A. Figura 1B mostra un coprioggetto adeguatamente lavati e asciugati. Se il precipitato, l&#39;intera procedura deve essere riavviato dal principio come il vetrino non è più utilizzabile. Dopo la formazione del idrogel e rivestimento con proteine ​​ECM durante la notte, le cellule possono essere seminati il ​​giorno successivo. Come mostra la Figura 2 mostra, vi è una netta differenza tra la diffusione delle cellule in idrogel rigido rispetto morbido. Come si può notare dalla colorazione falloidina nei fibroblasti embrionali di topo (MEF), le cellule diffuse in misura maggiore sul rigido rispetto a idrogel morbido. Infatti, la maggior parte delle cellule di legarsi a un idrogel morbido rimarrà compatto e collegare in modo meno efficiente. Anche se solo morfologia MEF è illustrato nella figura 2, la differenza nella cella diffusione è coerente tra diverse altre linee cellulari testate 11-12,14. I segreti del successo <ol><li> Una volta che la procedura viene avviata, è molto importante mantenere i coprioggetti con il lato &quot;reattiva&quot; e di tenere presente che parte è stato rivestito. </li><li> I guanti dovrebbero essere indossati in ogni momento durante la procedura di fornire un ambiente di lavoro che sia il più sterile possibile. </li><li> La maggior parte dei passi iniziali vengono eseguiti tra una cappa e il banco di laboratorio. Tutte le fasi successive alla rimozione glutaraldeide in eccesso dal coprioggetto devono essere eseguiti sotto una cappa di sicurezza biologica. </li><li> A causa delle differenze nella cella Spreading (Figura 2), si consiglia di seminare il doppio del numero di celle idrogel morbido rispetto a idrogel rigida. </li><li> Il processo di polimerizzazione AC è estremamente veloce. Per gli utenti prima volta, si può diminuire la quantità di APS o TEMED nella soluzione di prolungare il processo di polimerizzazione. Non cercare di preparare più di un paio di lamelle in una sola volta. </li><li> Negli studi del ciclo cellulare in cui è richiesta la sincronizzazione in G0, di solito siero affamare le cellule in coltura di plastica, le celle e trypsinize reseed li idrogel di adempimenti diversi in presenza di mitogeni (FBS e / o fattori di crescita). Questa procedura garantisce che la popolazione di partenza di G0-sincronizzata delle cellule è identico in tutti i campioni. Tuttavia, per molti studi di trasduzione del segnale, il necessario periodo di stimolo mitogeno potrebbe non essere sufficientemente lungo per consentire l&#39;attaccamento e la diffusione delle cellule. In questo caso, siero-affamare le cellule del idrogel e poi direttamente stimolarli con mitogeno. </li></ol><img src="/files/ftp_upload/2089/2089fig1a.jpg" alt="Figura 1a" /> Figura 1A. Impropriamente lavato coprioggetto. Dopo aggiunta di APTMS, coprioggetto è stato lavato per 1-2 minuti prima aggiunta della soluzione di glutaraldeide. Un precipitato si forma sul vetrino che non sia stata lavata secondo la procedura descritta in Procedura. <img src="/files/ftp_upload/2089/2089fig1b.jpg" alt="Figura 1b" /> Figura 1B. Adeguatamente lavati coprioggetto. Aggiunta seguito di APTMS, coprioggetto è stato lavato per tre volte 10 minuti ciascuno, prima della soluzione di glutaraldeide. Non precipitare forma sul vetrino. <img src="/files/ftp_upload/2089/2089fig2.jpg" alt="Figura 2" /> Figura 2. Morfologia delle cellule in idrogel di diversa rigidità. Fondata MEF sono state seminate su fibronectina rivestite di idrogel rigidità alto o basso per 9 ore. Dopo il periodo di incubazione, le cellule sono state fissate, permeabilizzate e colorati con FITC falloidina che si lega alla f-actina. MEF su gel di elevata rigidità mostra fibre stress e sono ben distribuiti rispetto a quelle seminate in idrogel bassa rigidità. Barra di scala = 50 micron. <img src="/files/ftp_upload/2089/2089fig3.jpg" alt="Figura 3" /> Figura 3. Rappresentante risultato PCR quantitativa del mouse livelli di mRNA della ciclina D1. Siero fibroblasti di topo affamato embrionali sono stati placcati su alta (3% di acrilammide) o bassa (0,3% di acrilammide) idrogeli rigidità e stimolati con 10% FBS per 9 ore. Dopo l&#39;estrazione di RNA, analisi real-time PCR quantitativa è stata effettuata per livelli di mRNA della ciclina D1 (normalizzato a 18S RNA). G0 rappresenta ciclina D1 mRNA da cellule quiescenti. Ciclina D1 aumentare significativamente i livelli di mRNA in idrogel rigido ma non su idrogel morbido. I dati sono media + / - SD di duplicare reazioni di PCR.

<ol>
	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Duprez, D. A., Cohn, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Lee, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+mechanical+properties+of+human+atherosclerotic+tissue+by+high-frequency+intravascular+ultrasound+imaging.+An+in+vitro+study.">Prediction of mechanical properties of human atherosclerotic tissue by high-frequency intravascular ultrasound imaging. An in vitro study.</a> Arterioscler Thromb. 12, 1-5 (1992).</li><li>Levental, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139, 891-906 (2009).</li><li>Paszek, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Samani, A., Zubovits, J., Plewes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+moduli+of+normal+and+pathological+human+breast+tissues:+an+inversion-technique-based+investigation+of+169+samples.">Elastic moduli of normal and pathological human breast tissues: an inversion-technique-based investigation of 169 samples.</a> Phys Med Biol. 52, 1565-1576 (2007).</li><li>Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+stiffness+in+regulating+cell+behavior.">The role of matrix stiffness in regulating cell behavior.</a> Hepatology. 47, 1394-1400 (2008).</li><li>Pelham, R. J., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proc. Natl. Acad Sci USA. 94, 13661-13665 (1997).</li><li>Klein, E. A., Yung, Y., Castagnino, P., Kothapalli, D., Assoian, R. K. <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,+cellular+tension,+and+cell+cycle+control.">Cell adhesion, cellular tension, and cell cycle control.</a> Methods Enzymol. 426, 155-175 (2007).</li><li>Klein, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle+control+by+physiological+matrix+elasticity+and+in+vivo+tissue+stiffening.">Cell-cycle control by physiological matrix elasticity and in vivo tissue stiffening.</a> Current Biology. 19, 1511-1518 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

Un elemento cruciale del processo di polimerizzazione idrogel è quello di evitare la formazione di bolle d&#39;aria che consentono alle cellule di legarsi al coprioggetto di vetro piuttosto che l&#39;ECM rivestite stesso idrogel. Ciò può essere evitato con cura di pipettaggio la soluzione di polimerizzazione dopo vortex e visivamente fare in modo che bolle d&#39;aria sono intrappolati nel gel. Noi consigliamo sempre la preparazione aggiuntiva &quot;a caldo&quot; coprioggetto e idrogeli per assicurare avere abbastanza...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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		<tbody>
		
<tr>
<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
</tr>
<tr>
<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
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studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

Rigidità del tessuto è un importante determinante della funzione cellulare, e cambiamenti nella rigidità dei tessuti sono comunemente associati con fibrosi, cancro e malattie cardiovascolari 1-11. Approcci tradizionali cellule biologiche per studiare la funzione cellulare coinvolgono le cellule coltura su un substrato rigido (piatti di plastica o vetro coprioggetto) che non possono spiegare l&#39;effetto di un ECM elastico o le variazioni di rigidezza ECM tra i tessuti. Per modellare condizioni in vivo nel rispetto dei tessuti in vitro, noi e altri usano ECM rivestite idrogel. Nel nostro laboratorio, l&#39;idrogel si basano su poliacrilammide che possono mimare la gamma di conformità dei tessuti biologicamente visto 12. &quot;Reattiva&quot; coprioggetto sono generati da incubazione con NaOH seguita da aggiunta di 3-APTMS. Glutaraldeide è usato per cross-link del 3-APTMS e il gel di poliacrilammide. Una soluzione di acrilamide (AC), bis-acrilamide (Bis-AC) e persolfato di ammonio è usato per la polimerizzazione degli idrogel. N-hydroxysuccinimide (NHS) è incorporato nella soluzione CA per proteine ​​crosslink ECM per l&#39;idrogel. A seguito di polimerizzazione del idrogel, la superficie del gel è rivestita con una proteina ECM di scelta, come fibronectina, vitronectina, collagene, ecc La rigidità di un idrogel può essere determinato da reologia o microscopia a forza atomica (AFM) e regolata variando la percentuale di AC e / o bis-CA nella soluzione di 12. In questo modo, la rigidità substrato può essere abbinata alla rigidità dei tessuti biologici che può anche essere quantificato mediante reologia o AFM. Le celle possono poi essere seminato in questi idrogel e colti sulla base delle condizioni sperimentali richieste. Imaging delle cellule e il loro recupero per l&#39;analisi molecolare è semplice. Per questo articolo, si definisce substrati morbidi come quelli che hanno moduli elastici (E) &lt;3000 Pascal e substrati rigidi / tessuti come quelli con E&gt; 20.000 Pascal.

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Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

L&#39;effetto di rigidità substrati sulla funzione cellulare può essere modellato<em&gt; In vitro</em&gt; Utilizzando idrogel di poliacrilamide di varia conformità.

Studiare gli effetti di rigidità Matrix sulla funzione cellulare utilizzando acrilamide a base di idrogeli

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

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21.9K Views.  University of Pennsylvania. L'effetto di rigidità substrati sulla funzione cellulare può essere modellato In vitro Utilizzando idrogel di poliacrilamide di varia conformità.

Video: Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Incapsulamento cellulare in idrogel 3D per l&#39;ingegneria tissutale

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Ricerca

Biologia

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

Misurare la rigidezza flessionale di cellule batteriche Utilizzando una trappola ottica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Identificare gli effetti delle mutazioni BRCA1 sulla ricombinazione omologa utilizzando cellule che esprimono endogeno wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analizzando la funzione di GTPasi Small Microiniezione di plasmidi in cellule epiteliali polarizzate

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Visualizzazione della funzione respiratoria mitocondriale utilizzando Citocromo C Ossidasi / succinato deidrogenasi (COX / SDH) Doppio etichettatura Istochimica

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

La creazione di ambienti definiti gassosi Per studiare gli effetti dell&#39;ipossia sulla<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Misurare gli effetti dei batteri su<em&gt; C. Elegans</em&gt; Comportamento utilizzando un saggio di ritenzione dell&#39;uovo

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Analisi della funzione Beta-cellulare usando la formazione immagine del calcio cella singola risoluzione in Zebrafish isolotti

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Un modello di coltura cellulare di adipociti per studiare l'impatto della modulazione di proteine e micro-RNA sulla funzione degli adipociti

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Analisi della funzione E3 Ubiquitina ligasi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...