This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President&#8217;s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.

1. Preparazione di soluzioni e Aliquote Hydrogel associati <ol><li> Preparazione della soluzione poliacrilammide gel precursore. <ol><li> Preparare soluzione di gel di poliacrilammide precursore miscelando acrilammide (A) (40% w / v, M r 71.08 g / mol), la bisacrylamide reticolante (B) (2% w / v, M r 154.17 g / mol), e de- acqua ionizzata nelle percentuali di volume specificati nella tabella 1. NOTA: Queste soluzioni possono essere preparati in grandi lotti e conservati a 4 ° C fino a diversi mesi. <ol><li> ATTENZIONE: L&#39;acrilamide è tossica su inalazione o ingestione, in particolare, se in polvere: così, utilizzare preferibilmente il 40% w / v soluzione per ridurre i rischi di tossicità. Maneggiare solo mentre indossa indumenti protettivi, come guanti, occhiali, e un camice da laboratorio. Conservare in un contenitore resistente alla luce, ben chiuso in frigorifero (&lt;23 ° C, e ben ventilato). </li><li> ATTENZIONE: Bis-acrilamide è tossica su inalazione o ingestione, in particolare,quando in polvere: così, utilizzare preferibilmente 2% w / v soluzione per ridurre i rischi di tossicità. Maneggiare solo mentre indossa indumenti protettivi, come guanti, occhiali, e un camice da laboratorio. Conservare in un contenitore resistente alla luce, ben chiuso in frigorifero (&lt;4 ° C, e ben ventilato). </li></ol></li></ol></li><li> Preparazione e utilizzo di N -Sulfosuccinimidyl-6- (4&#39;-azido-2&#39;-nitrophenylamino) esanoato (Sulfo-SANPAH, M r 492,40 g / mol) aliquote. ATTENZIONE: Sulfo-SANPAH provoca grave irritazione oculare; maneggiare con i guanti e la corretta protezione degli occhi o il viso. Conservare a -20 ° C alla ricezione e prima in aliquote. <ol><li> Per memorizzare sulfo-SANPAH: sciogliere Sulfo-SANPAH in dimethylsulfoxane (DMSO) a 50 mg / ml. Aliquota della soluzione di riserva in 50 provette micro da 20 ml per provetta. Freeze Flash in ghiaccio secco o in azoto liquido (un passaggio facoltativo, ma preferito per preservare la massima efficienza reticolante) e conservare a -80 ° C. NOTA: Le aliquote can essere conservati per alcuni mesi. </li><li> Per utilizzare solfo-SANPAH: scongelare brevemente un&#39;aliquota e diluire in 480 ml di acqua deionizzata. Utilizzare immediatamente. Sulfo-SANPAH idrolizza rapidamente in acqua: quindi prendere cautela per eseguire tutti i passaggi sopra in fretta. </li></ol></li><li> Preparazione di persolfato di ammonio (M r 228,18 g / mol) aliquote. NOTA: persolfato di ammonio provoca occhi, irritazione della pelle e delle vie respiratorie. Indossare dispositivi di protezione adeguati durante la manipolazione. Maneggiare persolfato di ammonio in polvere in una cappa. Conservare la polvere in un luogo asciutto e ben ventilato. Una volta aliquotati, la soluzione diluita può essere utilizzato sul banco di lavoro. <ol><li> Sciogliere persolfato di ammonio in acqua deionizzata per ottenere una concentrazione finale persolfato di ammonio del 10% w / v. Aliquota e conservare a -20 ° C. Scongelare immediatamente prima dell&#39;uso. </li></ol></li><li> Preparazione della soluzione di collagene di tipo I. <ol><li> Preparare 0,2 mg / ml soluzione di collagene diluendo il magazzino in modoluzione in 1x tampone fosfato salino (PBS) pH 7,4. Conservare brevemente ghiaccio o utilizzare immediatamente dopo la diluizione. </li></ol></li></ol> 2. Idrogel Preparazione (fare riferimento alla figura 1) <ol><li> Preparazione di vetrini idrofobici. <ol><li> Mettere alcune gocce di una soluzione idrofobico su una lastra di vetro e utilizzare carta velina per diffondere attraverso la superficie. Lasciate asciugare e pulire con un fazzolettino di carta di nuovo per uniformare il rivestimento idrofobo. NOTA: soluzione idrofobico Conservare in un armadio infiammabile a temperatura ambiente. Indossare indumenti protettivi durante la manipolazione. Lavorare in un ambiente ben ventilato. </li></ol></li><li> Preparazione del supporto plastico flessibile. <ol><li> Tagliare il supporto di plastica flessibile per adattarsi alle dimensioni della lastra di vetro idrorepellente rivestita. </li><li> Contrassegnare lato idrofoba del supporto plastico flessibile leggermente graffiare la superficie con un attrezzo appuntito come un bisturi. NOTA: Una volta che il gel - che è completamente trasparente - asciuga, I graffi aiuteranno a distinguere da che parte il supporto di plastica flessibile contiene il gel. NOTA: Il supporto di plastica flessibile ha un idrofobico e un lato idrofila. Una volta depositato, poliacrilammide aderisce in modo permanente al lato idrofila del supporto plastico che facilita la successiva manipolazione idrogel. </li></ol></li><li> Gel Preparazione (esempio volumi sono indicati per soluzioni gel precursori 5 ml). NOTA: 5 ml sarebbero sufficienti a produrre ~ 40 gel quando lo spessore iniziale gel è di 0,5 mm. Più gel possono essere prodotti se lo spessore gel viene ridotta. <ol><li> Inserire 4972,5 ml di soluzione di precursore di poliacrilammide concentrazione finale desiderata (Tabella 1) in una provetta conica da 50 ml. Mettere in una camera di degasaggio per 30 minuti con il tappo aperto. NOTA: ossigeno agisce come una trappola radicali liberi e, quando presente, inibisce la polimerizzazione. </li><li> Aggiungere 25 ml di 10% w / v di ammonio persolfato (vedi punto 1.3) per ottenere una finaleammonio persolfato concentrazione dello 0,05%. </li><li> Aggiungere 2,5 ml di N, N, N &#39;, N&#39;-tetrametiletilendiammina (TEMED, M r 116.24 g / mol) alla soluzione di gel degasato per ottenere una concentrazione finale di 0,5 TEMED%. NOTA: Conservare TEMED in un armadio infiammabile a temperatura ambiente. Maneggiare in una cappa indossando i dispositivi di protezione adeguati. Tenere ben chiuso sotto gas inerte, come è altamente dell&#39;aria e sensibile all&#39;umidità. </li><li> Mescolare delicatamente la soluzione pipettando su e giù 3 - 5 volte. Non vortice per evitare la diffusione di ossigeno nella soluzione di gel precursore. </li><li> Pipettare la soluzione di gel sul lato idrofila del supporto plastico flessibile e panino con idrofobico rivestita vetrino. Separate le due slitte con distanziali silicone di spessore desiderato (ad esempio 0,5 mm). Lasciare una piccola quantità (~ 100 ml) di soluzione di precursore di polimero nella provetta conica da 50 ml di utilizzare come indicatore di gelificazione. NOTA:Qualsiasi spacer potrebbe essere utilizzato. Per esempio, una singola striscia di Parafilm dà spessore gonfio gel finale di 100 - 120 micron. </li><li> Posizionare un altro lastra di vetro in cima al flessibile panino supporto / gel plastica per accertare una superficie piana idrogel sulla polimerizzazione. </li><li> Lasciare polimerizzare il gel per 45 minuti, anche se meno tempo può essere utilizzato per i gel di maggiore% in peso, se desiderato. Per verificare che il gel ha polimerizzato, osservare la soluzione rimanente nel tubo conico da 50 ml; se la soluzione rimanente è gelificata, allora è probabile che gelificazione sulla lastra di vetro è stata avviata pure. Si noti tuttavia che l&#39;apertura anticipata dello stampo impedirà completa polimerizzazione. </li><li> Una volta che il gel si forma, staccare il supporto plastico flessibile con il gel di poliacrilammide covalentemente attaccato sulla parte superiore, e impostare gel-side-up asciugare all&#39;aria. NOTA: convezione o essiccazione calore possono anche essere utilizzati per accelerare questo passaggio. Una volta essiccato sul supporto di plastica flessibile, il gel può essere negoziod tempo indeterminato. <ol><li> Segna tutti i punti scoperti del supporto plastico flessibile, dove gel di poliacrilammide non formano a causa di bolle. Mark mentre il gel è ancora idratata quanto ciò fondono con il supporto plastico flessibile quando il gel è asciutto. </li></ol></li></ol></li></ol> 3. multipozzetto gruppo piastra, Collagene Coating, e sterilizzazione <ol><li> Assieme piastra multipozzetto. <ol><li> Una volta asciugato, tagliato gel PA in forme desiderate. <ol><li> Per una piastra a 96 pozzetti, utilizzare un buco pugno pesante, con un diametro di circa 6 mm. Usare un taglierino pesante per tagliare gel in forme quadrate o rettangolari. In alternativa, usare le forbici. </li></ol></li><li> Preparare circa 500 ml di PDMS per piastra da 96 pozzetti secondo le istruzioni del produttore. Per incollare i gel al fondo di una piastra multipozzetto, posizionare una piccola goccia (~ 5 microlitri) di polidimetilsilossano (PDMS) al centro della ogni pozzetto. Utilizzando pinze, collocare uno polyacrylamide gel in ogni lato supporto plastico bene, flessibili verso il basso. Per consentire PDMS curare, lasciare la piastra montata a 37 ° C per un minimo di 4 ore. </li></ol></li><li> Rivestimento Collagene di gel di poliacrilammide. <ol><li> Con una pipetta di trasferimento, mettere una piccola quantità (7-8 ml) di solfo-SANPAH (vedi punto 1.2.2) in ogni pozzetto e turbolenza da un lato all&#39;altro per rivestire la superficie del gel in modo uniforme. Lavoro prontamente dal solfo-SANPAH non è stabile in acqua. </li><li> Posizionare la piastra ben sotto una lampada UV ad alta intensità (intensità = 37 mW, λ = 302-365 nm) per 5 min. Sciacquare i gel con PBS per rimuovere l&#39;eccesso solfo-SANPAH. </li><li> Pipettare 50 ml di 0,2 mg / ml di collagene di tipo I soluzione (vedi punto 1.4) in ogni pozzetto. Lasciare la piastra coperta a temperatura ambiente per almeno 2 ore o O / N a 4 ° C. NOTA: Per accelerare il processo di verniciatura collagene, una pipetta di trasferimento può essere utilizzata per aggiungere la soluzione di collagene. Tipicamente, 1 - 2 gocce di soluzione deve essere sufficiente per completarely coprire la superficie del gel. </li><li> Lasciare la piastra bene a temperatura ambiente per almeno 2 ore per consentire il collagene legame. </li><li> Risciacquare con PBS per rimuovere la soluzione di collagene in eccesso e sterilizzare ai raggi UV (λ = 200 nm) in una cappa coltura tissutale per 2 ore. </li><li> Mettere a bagno i gel in terreno completo (fare riferimento alla sezione 4.1 per mezzo di composizione) O / N per idratare ed equilibrare. Utilizzare per la cella di semina immediatamente o conservare in frigorifero per un massimo di 2 giorni. </li></ol></li></ol> 4. Semina cellulare su PA Rigidità Assay NOTA: Sebbene tipico per linee comuni di cellule di mammifero, il protocollo descritto in questa sezione è specificamente utilizzato con il cancro al seno linea cellulare MDA-MB-231 (vedi figure 4 e 5). <ol><li> Raccogliere cellule dal pallone di coltura tissutale da esposizione al 5% tripsina / EDTA per 5 min a 37 ° C. Utilizzare ~ 80 ml ​​di tripsina / EDTA per ogni cm 2 di cultura dell&#39;area pallone; per esempio, utilizzare 2 ml di tripsina / EDTA fora T25 pallone di coltura cellulare. Risospendere le cellule raccolte in mezzo completo addizionato con siero fetale bovino 10% (FBS) e 1% di penicillina / streptomicina a una concentrazione cellulare finale desiderata. Tenendo conto delle cellule tempo di raddoppio e la lunghezza del tempo le cellule saranno seminate sul dosaggio, scegliere concentrazione cellulare sub-confluenti appropriata. Assicurarsi che media aggiunto è sufficiente per immergere completamente il idrogel. NOTA: Poiché i idrogel sono stati pre-equilibrata nei media (fare riferimento al punto 3.2.6) 100 Volume ml dovrebbe essere sufficiente. Tipici volumi medi utilizzati per piastre a pozzetti multipli devono essere sufficienti in quanto i gel sono stati pre-equilibrata in media nel passaggio precedente. NOTA: Il test sarebbe appropriato per qualsiasi tipo di cellula attaccamento-dipendente. <ol><li> Contare il numero di cellule al microscopio invertito utilizzando un emocitometro. Carico 10 ml di sospensione cellulare in ogni porto emocitometro e la media del numero di celle da almeno 8 quadranti. Per ottenere ilconcentrazione cellulare finale, moltiplicare il numero di celle da 10 4. NOTA: I migliori risultati di conteggio delle cellule si ottengono quando il numero di cellule in ogni quadrante emocitometro è 20 - 50. </li></ol></li><li> Coltivare le cellule sul test rigidità PA in un incubatore umidificato a 37 ° C e 5% di CO 2. Cambiare i media ogni 2 - 3 giorni. Raccogliere cellule quando necessario dall&#39;esposizione al 5% tripsina / EDTA a 37 ° C per 5 min. Utilizzare ~ 80 ml ​​di tripsina / EDTA per cm 2 di pallone di coltura tissutale. Durante tutte le fasi di manipolazione cellulare, prestare particolare attenzione a non disturbare la superficie di idrogel: aspirato o medio pipetta leggermente inclinando la piastra pozzetti lateralmente e toccando la punta della pipetta sulla parete laterale di ogni bene, al contrario di toccare l&#39;idrogel. NOTA: Ad eccezione facendo particolare attenzione a non danneggiare la superficie idrogel durante la movimentazione coltura tissutale normale, le cellule seminate su dosaggio rigidità può essere manipolato nello stesso modo come se fossero seminate suad una piastra multipozzetto regolare. </li></ol> 5. Imaging di celle seminate su PA Rigidità Assay <ol><li> Celle di immagine direttamente sul test rigidità PA. NOTA: Qualsiasi microscope - invertito, fluorescenza o confocale, può essere utilizzata per l&#39;imaging cellulare. NOTA: Il supporto di plastica flessibile utilizzato per costruire il test di rigidità è completamente trasparente e non possiede autofluorescenza o interferire con l&#39;imaging cellulare. Tuttavia, anche se il supporto flessibile di plastica è trasparente, capacità di imaging saranno limitati dalla distanza di lavoro dell&#39;obiettivo. Il supporto di plastica flessibile ha uno spessore di 0,23 mm e una distanza di lavoro tipica di un obiettivo 10X è ~ 4 mm bruscamente decrescente per ingrandimenti maggiori. <ol><li> Per l&#39;imaging cellulare dal vivo, posizione PA rigidità test in portatarga microscopio e immagine. Tenere sessioni di imaging in 2 ore, o utilizzare un microscopio equipaggiato con una camera climatica per tempi più lunghi di imaging. </li><li> Quando im cellulare dal vivol&#39;invecchiamento non è l&#39;obiettivo, fissare le cellule in soluzione di formaldeide 4% integrato con 0,1% di detergente. Immergere le cellule in fissante a temperatura ambiente per un minimo di 2 ore. Risciacquare con PBS due volte. Smaltire i rifiuti fissativo in un contenitore per rifiuti designato. NOTA: Le celle possono essere esposte immediatamente o conservate immersi in PBS a 4 ° C fino a 2 settimane. ATTENZIONE: La formaldeide è tossica su inalazione e contatto. Maneggiare con guanti in una cappa. NOTA: il protocollo di fissazione delle cellule deve essere scelto in base ai protocolli di colorazione e di trattamento delle cellule successive, in quanto alcuni fissativi danneggiano alcune proteine ​​cellulari. </li><li> Per l&#39;imaging fluorescente, macchia cellule con colorazione cella desiderata direttamente nel piatto pozzetti fisso. Immagine immediatamente. </li></ol></li></ol>

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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-cell+mechanical+communication+through+compliant+substrates.">Cell-cell mechanical communication through compliant substrates.</a> Biophysical journal. 95 (12), 6044-6051 (2008).</li><li>Fischer, R. S., Myers, K. A., Gardel, M. L., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stiffness-controlled+three-dimensional+extracellular+matrices+for+high-resolution+imaging+of+cell+behavior.">Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior.</a> Nature protocols. 7 (11), 2056-2066 (2012).</li><li>Quinlan, A. M., Billiar, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+role+of+substrate+stiffness+in+the+persistence+of+valvular+interstitial+cell+activation.">Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation.</a> Journal of Biomedical Materials Research Part A. 100 (9), 2474-2482 (2012).</li><li>Zustiak, S. P., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrolytically+degradable+poly(ethylene+glycol)+hydrogel+scaffolds+with+tunable+degradation+and+mechanical+properties.">Hydrolytically degradable poly(ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties.</a> Biomacromolecules. 11 (5), 1348-1357 (2010).</li><li>Chrambach, A., Rodbard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+gel+electrophoresis.">Polyacrylamide gel electrophoresis.</a> Science. 172 (3982), 440-451 (1971).</li><li>Lin, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensing+of+substrate+thickness.">Mechanosensing of substrate thickness.</a> Physical review. 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The authors have nothing to disclose.

Gel di poliacrilammide, originariamente sviluppati per elettroforesi, 28 sono oggi utilizzati di routine come substrati di coltura cellulare per studiare gli effetti di substrato rigidità sulla morfologia delle cellule, la motilità e la comunicazione 3,24,29 tra le altre caratteristiche cellulari. Poliacrilammide consente la manipolazione del substrato rigidità che comprende la rigidità di tutti i tessuti molli del corpo (0,3 - 300 kPa) 1 con una semplice variazione della concentrazi...

La maggior parte dei tessuti del corpo sono materiali viscoelastici morbidi con un modulo di Young che vanno da 0,1 kPa per cervello a 100 kPa per cartilagine morbida, ma, più in ricerca sulle cellule vitro è condotta su coltura tissutale polistirene (TCP), che ha un modulo di ~ 1 GPa . 1 Questa mancata corrispondenza rigidità influenza notevolmente il modo in cui le cellule rispondono al loro ambiente. Un crescente corpo di ricerca è quindi dedicata alla chiarire l&#39;effetto del substrato rigidità sul destino di vari tipi cellulari, comprese le cellule staminali 2,3. 4 Come risultato, più idrogel sono stati sviluppati per aiutare nella comprensione della cella rigidità-dipendente biologia compresi poliacrilammide (PA), 5-7 polietilene glicole (PEG), 8,9 polidimetilsilossano (PDMS), 10 e alginato. 11 Mentre la prova che substrato rigidità ha un impatto sostanziale sul destino delle cellule è in crescita, la maggior parte degli studi sono condotti su scala ridotta con un piccolo numero di samples. Sistematiche, studi multidimensionali sull&#39;effetto di substrato rigidità per una serie di tipi cellulari o condizioni ambientali sono rare. 12 Sono state sviluppate diverse tecnologie idrogel high-throughput promettenti, tra cui microarrays basati PEG, 13 dispositivi microfluidici per la produzione di microsfere di agarosio idrogel, 14 o micro e nano-aste in cui la rigidità è modulata dal diametro e altezza delle microrods. 15 Tuttavia , le tecnologie per preparare questi substrati sono sofisticati e disponibili per il numero limitato di laboratori. Gran parte della ricerca che coinvolge rigidità modulato le risposte delle cellule utilizza poliacrilammide (PA) gel che non sono solo poco costoso e semplice da implementare, ma mostrano anche una gamma fisiologicamente rilevanti di modulo di Young, vale a dire 0,3 -. 300 kPa 16-22 Tuttavia, i metodi esistenti per fabbricare PA gel per colture cellulari molto lavoro e, conseguentemente, di preparazioneared in piccoli lotti. Alcune delle difficoltà associate alla preparazione di gel PA come substrati cellulari derivano dal requisito che i gel devono essere preparati: 1) in assenza di ossigeno per permettere la completa polimerizzazione, 2) con una superficie piana e liscia per consentire cellulare uniforme attaccamento e diffusione, e 3) fissata in modo al fondo del piatto di coltura cellulare per prevenire galleggiante. Diversi gruppi hanno tentato di produrre gel PA per colture cellulari in grandi lotti. Semler et al. Fogli spessi preparati dei gel PA che erano poi &quot;tagliata&quot; con un perforatore e posizionati in piastre da 96 pozzetti. 23 Tuttavia, questo metodo è limitato a gel rigide, cioè,&gt; 1 kPa nel modulo di Young, perchè più morbido gel sono &quot;sticky&quot;, difficile da tagliare, e facilmente danneggiati. Mih et al. Sviluppato una tecnica più sofisticata che permette i gel da polimerizzare direttamente in una piastra multipozzetto fondo di vetro. <sup&gt; 6 Questo è stato ottenuto versando le soluzioni di gel in lastre di vetro-bottom funzionalizzate e formando gel da &quot;sandwich&quot; di loro con un allineamento coprioggetti personalizzato. 6 Anche se, lievi effetti di bordo molto promettenti sono stati ancora osservati con questa tecnica. Inoltre, la tecnica richiede una matrice custom-designed non immediatamente accessibili a molti laboratori e costose piastre a pozzetti multipli con fondo di vetro. Questo documento descrive un modo semplice e poco costoso da montare gel PA in una piastra multipozzetto che potrebbe facilmente essere adottata da qualsiasi laboratorio. Qui, un supporto plastico flessibile è utilizzato, che ha due lati - uno idrofoba, che è repellente per gel PA, e uno idrofila, che si lega covalentemente il gel PA sulla deposizione. Una volta fogli di gel PA vengono depositati e permanentemente affissa al supporto plastico flessibile, consente la movimentazione gel di qualsiasi spessore o rigidità e taglio in qualsiasi forma desiderata. Questo approach non solo produce custom &#39;vetrini&#39; di plastica in formati non altrimenti disponibili in commercio, ma evita anche la necessità di pre-trattare le superfici di vetro, sia vetrini o pozzetti di costose lastre di pozzetti con fondo di vetro, con una soluzione PA vincolante, che è un noioso e un passo che richiede tempo. Infine, uniformi fogli gel PA possono essere preparati in grandi lotti e de-idratato conservati per alcuni mesi. In sintesi, il saggio qui presentato è un miglioramento rispetto ai metodi esistenti in diversi aspetti. In primo luogo, il processo di assemblaggio piastra multipozzetto è efficiente, e il costo complessivo dei materiali necessari è basso. In secondo luogo, gli idrogel sono prodotte in grandi lotti in un unico film gel omogeneo. Infine, solo materiali che sono commercialmente disponibili sono richiesti. L&#39;utilità del saggio è illustrato esplorare l&#39;effetto di substrato rigidità sulla morfologia cellulare e zona diffusione.

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Reagents</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">40% Acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0140</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">2% Bis-acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Ammonium Persulfate</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-07000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">TEMED</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T9281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Sulfo-SANPAH</td>
			<td style="width:200px;">Thermo Scientific</td>
			<td style="width:187px;">22589</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Collagen Type 1, from Rat tail, 3.68 mg/mL</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:187px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:200px;">Fisher Scientific&#160;</td>
			<td style="width:187px;">BP231-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Hydrophobic solution - Repel Silane&#160;</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">17-1332-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">PBS (1x), pH 7.4</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:303px;">Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td>
			<td style="width:200px;">Elsworth Adhesives</td>
			<td style="width:187px;">3097358-1004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tyrpsin/EDTA (10x)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">44174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RPMI-1640 Medium (1x)</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30027-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30073-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Penicillin Streptomycin</td>
			<td style="width:200px;">MP Biomedicals</td>
			<td style="width:187px;">1670046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent - Triton-X</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Formaldehyde 37% Solution</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">A2153</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">BSA-based cell adhesion blocking kit - ECM Cell Adhesion Array Kit</td>
			<td style="width:200px;">Chemicon International</td>
			<td style="width:187px;">ECM540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">flexible plastic support - GelBond PAG Film for Polyacrylamide Gels</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">309819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Glass Plates</td>
			<td style="width:200px;">Slumpys</td>
			<td style="width:187px;">GBS4100SFSL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">50 mL Conicals</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">3181345107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">15 mL Conicals</td>
			<td style="width:200px;">FALCON</td>
			<td style="width:187px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Micro centrifuge tubes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2 mL: 02681258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">96-well plate (flat bottom)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">12565501</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:303px;">Disposable Pipettes (1 mL, 2mL, 5mL, 10mL, 25 mL, 50mL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Glass Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">5 3/4&#34;: 1367820A, 9&#34;:136786B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Tips (1-200uL, 101-1000uL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2707509</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Plastic Standard Disposable Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:187px;">13-711-9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Parafilm</td>
			<td style="width:200px;">PARAFILM&#160;</td>
			<td style="width:187px;">PM992</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Powder Free Examination Gloves</td>
			<td style="width:200px;">Quest</td>
			<td style="width:187px;">92897</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td>
			<td style="width:200px;">Grace Bio-Labs</td>
			<td style="width:187px;">JTR-S-0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Large/non-disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Light and Flourescent Microscope (Axiovert 200M)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">3820005619</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope Software</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">AxioVision Rel. 4.8.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">UV oven</td>
			<td style="width:200px;">UVITRON</td>
			<td style="width:187px;">UV1080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum chamber/degasser</td>
			<td style="width:200px;">BelArt</td>
			<td style="width:187px;">999320237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum pump for degasser</td>
			<td style="width:200px;">KNF Lab</td>
			<td style="width:187px;">5097482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tissue Culture Hood</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-425-600</td>
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			<td height="21" style="height:21px;width:303px;">Chemical Fume Hood</td>
			<td style="width:200px;">KEWAUNEE</td>
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			<td height="21" style="height:21px;width:303px;">Inverted Microscope (Axiovert 25)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">663526</td>
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			<td height="21" style="height:21px;width:303px;">Incubator</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-8500</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Aid</td>
			<td style="width:200px;">Drummond Scientific Co.</td>
			<td style="width:187px;">P-76864</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemacytometer</td>
			<td style="width:200px;">Bright-Line</td>
			<td style="width:187px;">383684</td>
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simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Poliacrilammide (PA) idrogel sono ampiamente usati per testare le risposte delle cellule rigidità-dipendente. 17,24 Miscelando varie concentrazioni di acrilammide (A) e bis-acrilamide (B) si possono fare gel PA che abbracciano la gamma di maggior rigidità tessuti molli in il corpo - 0,3 -.. 300 modulo di kPa giovane 1 Tuttavia, la preparazione di gel di poliacrilammide è noioso e che richiede tempo, spesso limitando la loro utilità in applicazioni &quot;high throughput&quot; come per lo screeni...

Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Semplice Poliacrilammide-based a più pozzetti Rigidità Assay per lo Studio della rigidezza-dipendenti le risposte delle cellule

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

simple-polyacrylamide-based-multiwell-stiffness-assay-for-study

Research

JoVE Journal

Bioengineering

19.4K Views.  Saint Louis University. Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Video: Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Imaging bioluminescenza per la valutazione delle risposte immunitarie A seguito di impianto di tessuto cardiaco Composti (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

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Bioingegneria

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Un semplice Hanging goccia Cultura protocollo cellulare per la generazione di sferoidi 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotipiche Collagene I test: una piattaforma malleabile per valutare Comportamento cella in un 3-dimensionale Contesto

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

Saggio basato su cellule di calcio per le medie e High Throughput Screening del PRT Funzioni canale utilizzando FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

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

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

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

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

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

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

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Aspirazione Micropipetta di celle substrato-attached per Stima rigidità cellulare

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Utilizzando Chips microfluidica per l&#39;imaging dal vivo e studio delle risposte lesioni in<em&gt; Drosophila</em&gt; Larve

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparazione del 3D collagene Gel e microcanali per lo studio delle interazioni 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Un metodo semplice e scalabile di fabbricazione per dispositivi elettronici organici sui tessili

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Analisi 3D delle risposte multi-cellulari ai gradienti chemoattractant

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...