This study was supported by a Merit Award from the U.S. Department of Veterans Affairs, and an NIH grant R01CA1385282522717 to RW.

1. Preparazione dei materiali e reagenti <ol><li> Etichettare ciascun pozzetto di una coltura di tessuti trattati 6 pozzetti in modo appropriato per ogni linea cellulare o condizione sotto inchiesta. </li><li> Preparare 2x terreno di coltura cellulare sciogliendo 1 g di polvere media e 0,2 g di bicarbonato di sodio in acqua deionizzata fino ad un volume finale di 50 ml. </li><li> Passare questo mezzo attraverso un filtro da 0,2 micron per sterilizzare. </li><li> Aggiungere componenti aggiuntivi necessari per il normale coltura della linea cellulare di interesse. Ad esempio, crescere CMT linea 167 cellule in terreno RPMI 1640 supplementato con 10% FBS e 1% di penicillina / streptomicina soluzione. Medio Riscaldare a 37 ° C in bagno d&#39;acqua calda prima dell&#39;uso. </li><li> Preparare 1x terreno di coltura cellulare a parte come si farebbe per la coltura delle cellule normali della linea di cellule di interesse. </li><li> Preparare 1% nobile agar aggiungendo 1 g di agar nobile a 100 ml di acqua deionizzata. NOTA: agar Noble non si dissolvono completamente con l&#39;agitazione da solo. </li><li> Preparare0,6% nobile agar aggiungendo 0,6 g di agar nobile a 100 ml di acqua deionizzata. Entrambe le soluzioni agar possono essere realizzati in 100 ml bottiglie di vetro con coperchi richiudibili per la conservazione a lungo termine. </li><li> Autoclave le miscele di agar nobili per sterilizzare. Queste miscele possono essere fatte in anticipo e conservati a 4 ° C, ma devono essere riscaldati nuovamente al momento dell&#39;esperimento fino agar si è completamente dissolto. </li><li> Preparare la soluzione di cloruro di tetrazolio nitroblue facendone / ml di soluzione 1 mg in PBS 1x (8 g di NaCl, 0,2 g di KCl, 1,44 g di Na 2 HPO 4, e KH 2 PO 4 0,24 g di H 2 O a volume finale di 1000 ml ). Questo verrà utilizzato alla fine dell&#39;esperimento per colorare le colonie. </li></ol> 2. placcatura di strato inferiore di Agar <ol><li> Allentare il tappo sulla bottiglia del 1% nobile agar e forno a microonde per circa 1-2 min. Mentre il riscaldamento in un forno a microonde, monitorare attentamente la soluzione per evitare bollente. Si continua il riscaldamento, mentre la miscelazione in modo intermittente,fino agar è completamente sciolto e la soluzione è limpida. NOTA: Usare guanti resistenti al calore per gestire pallone dopo il riscaldamento. In caso contrario si potrebbe provocare ustioni o lesioni gravi. </li><li> Posizionare soluzione agar fuso e pre-riscaldato terreno di coltura 2x in un secchio di ghiaccio riempito con acqua calda (42 ° C). Collocare anche una provetta conica da 50 ml in un portaprovette nel secchio di ghiaccio con acqua calda. Trasferimento secchio per cappa di coltura cellulare per le fasi successive. </li><li> Per lo strato inferiore di agar, avrete bisogno di 1,5 ml di una miscela di agar e medie per pozzetto di una piastra da 6 pozzetti. </li><li> Per assicurare un&#39;adeguata quantità di miscela, preparare un totale di 12 ml per ogni piastra da 6 pozzetti. </li><li> Inizia con l&#39;aggiunta di 6 ml di terreno di coltura per il tubo conico da 50 ml e poi 6 ml di soluzione di nobile agar 1%. </li><li> Invertire il tubo conico più volte per mescolare. Lavorare a passo svelto si evita il rapido indurimento di agar molle. </li><li> Elaborare circa 5,5 ml di composto in un 5 ml pi sierologicapipetta. </li><li> Lasciare le bolle d&#39;aria a salire al top della colonna pipetta prima di depositare 1,5 ml di questa miscela in ogni pozzetto. Usare cautela per evitare il deposito di eventuali bolle d&#39;aria nei pozzetti della piastra. </li><li> Coprire le piastre e lasciare la miscela agar per solidificare a temperatura ambiente, in cappuccio coltura cellulare, per 30 min. </li></ol> 3. Placcatura Lo strato superiore di cellule contenenti Agar <ol><li> Una volta che lo strato inferiore di agar è solidificato, iniziare la preparazione dello strato superiore. </li><li> In primo luogo, le cellule raccolto mediante tripsinizzazione e diluire li 1: 5 in terreno di coltura (ad esempio per 1 ml di tripsina, aggiungere 4 ml di terreno) in una provetta conica da 15 ml. </li><li> Contare le cellule e calcolare il numero di cellule necessarie per pozzetto per preparare una sospensione cellulare finale in questo momento. Questo numero può variare a seconda del tipo di cellula. Utilizzare 5.000 cellule / pozzetto come punto di partenza e regolare come necessario. Per questo numero di cellulare, si dovrebbe preparare una sospensione di cellule di 6667 cellule / ml (cioè ogni well riceverà 0,75 ml di questa sospensione e 0,75 ml di agar per un volume totale di 1,5 ml; la concentrazione di cellule sarà diluito 1: 2 per un conteggio finale di cellule totale di 5.000). </li><li> Il volume di sospensione cellulare necessaria per pozzetto di una piastra da 6 pozzetti sarà nuovamente 1,5 ml. Preparare sospensione cellulare supplementare per un totale di 12 ml per piastra da 6 pozzetti. </li><li> Sciogliere soluzione agar 0,6% in un forno a microonde come sopra e riporre in secchiello per il ghiaccio contenente acqua calda con un tubo da 50 ml in un supporto tubo e la sospensione cellulare finale dal punto 3.3. </li><li> Trasferire il secchiello del ghiaccio con il fuso 0,6% agar per cappa di coltura cellulare per le fasi successive. </li><li> Mescolare 0,6% agar e sospensione di cellule in un rapporto 1: 1, la preparazione di un volume totale di 12 ml per piastra da 6 pozzetti. 1,5 ml saranno necessari per bene, ma in più devono essere effettuate come sopra. </li><li> Pipettare 6 ml di sospensione cellulare nel tubo conico da 50 ml. </li><li> Poi aggiungere 6 ml di 0,6% agar al tubo. NOTA: La temperatura di questa miscela deve esseretenuto circa 42 ° C per evitare il rapido indurimento e per massimizzare la sopravvivenza cellulare. </li><li> Lavorando in fretta, pipetta questa miscela 2-3x per distribuire le cellule, poi elaborare 5,5 ml di composto in un 5 ml pipetta sierologica. </li><li> Consenti eventuali bolle d&#39;aria a salire a cima della colonna pipetta prima di depositare 1,5 ml di questa miscela in ogni pozzetto. Usare cautela per evitare il deposito di eventuali bolle d&#39;aria nei pozzetti della piastra. </li><li> Lasciare miscela cellulare / agar per solidificare a temperatura ambiente, in cappuccio coltura cellulare, per 30 minuti prima di mettere in un 37 ° C coltura cellulare incubatore umidificato. </li><li> Il tempo necessario per un&#39;adeguata formazione di colonie varia per ogni linea cellulare, tipicamente circa 21 giorni. </li><li> Uno strato di terreno di coltura deve essere mantenuta al di sopra dello strato superiore di agar per prevenire l&#39;essiccamento. 100 pl di mezzo aggiunto due volte a settimana è sufficiente per questo scopo. </li></ol> 4. Colorazione le piastre e il conteggio delle colonie <ol><li> Cellule Macchia con l&#39;aggiunta di 200 _6; l di soluzione di cloruro nitroblu tetrazolio per pozzetto e incubando le piastre per una notte a 37 ° C. </li><li> Una volta che le colonie sono macchiati, scattare fotografie di pozzi con un imager e contare le colonie utilizzando il software di analisi delle immagini. </li></ol>

<ol>
	<li>Puck, T. T., Marcus, P. I., Cieciura, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+growth+of+mammalian+cells+in+vitro;+growth+characteristics+of+colonies+from+single+HeLa+cells+with+and+without+a+feeder+layer.">Clonal growth of mammalian cells in vitro; growth characteristics of colonies from single HeLa cells with and without a feeder layer.</a> J Exp Med. 103, 273-283 (1956).</li><li>Taddei, M. L., Giannoni, E., Fiaschi, T., Chiarugi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoikis:+an+emerging+hallmark+in+health+and+diseases.">Anoikis: an emerging hallmark in health and diseases.</a> The Journal of pathology. 226, 380-393 (2012).</li><li>Wend, P., Holland, J. D., Ziebold, U., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+in+stem+and+cancer+stem+cells.">Wnt signaling in stem and cancer stem cells.</a> Semin Cell Dev Biol. 21, 855-863 (2010).</li><li>Reya, T., 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=A+role+for+Wnt+signalling+in+self-renewal+of+haematopoietic+stem+cells.">A role for Wnt signalling in self-renewal of haematopoietic stem cells.</a> Nature. 423, 409-414 (2003).</li><li>Klaus, A., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+and+its+impact+on+development+and+cancer.">Wnt signalling and its impact on development and cancer.</a> Nat Rev Cancer. 8, 387-398 (2008).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+in+development+and+disease.">Wnt/beta-catenin signaling in development and disease.</a> Cell. 127, 469-480 (2006).</li><li>Takahashi-Yanaga, F., Kahn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Wnt+signaling:+can+we+safely+eradicate+cancer+stem+cells.">Targeting Wnt signaling: can we safely eradicate cancer stem cells.</a> Clin Cancer Res. 16, 3153-3162 (2010).</li><li>De, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/Ca2++signaling+pathway:+a+brief+overview.">Wnt/Ca2+ signaling pathway: a brief overview.</a> Acta Biochim Biophys Sin (Shanghai). 43, 745-756 (2011).</li><li>Tennis, M. A., Vanscoyk, M. M., Wilson, L. A., Kelley, N., Winn, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+of+Wnt7a+is+modulated+by+DNMT1+and+cigarette+smoke+condensate+in+non-small+cell+lung+cancer).">Methylation of Wnt7a is modulated by DNMT1 and cigarette smoke condensate in non-small cell lung cancer).</a> PLoS One. 7, (2012).</li><li>Winn, R. A., 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=Restoration+of+Wnt-7a+expression+reverses+non-small+cell+lung+cancer+cellular+transformation+through+frizzled-9-mediated+growth+inhibition+and+promotion+of+cell+differentiation.">Restoration of Wnt-7a expression reverses non-small cell lung cancer cellular transformation through frizzled-9-mediated growth inhibition and promotion of cell differentiation.</a> J Biol Chem. 280, 19625-19634 (2005).</li><li>Winn, R. A., 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=Antitumorigenic+effect+of+Wnt+7a+and+Fzd+9+in+non-small+cell+lung+cancer+cells+is+mediated+through+ERK-5-dependent+activation+of+peroxisome+proliferator-activated+receptor+gamma.">Antitumorigenic effect of Wnt 7a and Fzd 9 in non-small cell lung cancer cells is mediated through ERK-5-dependent activation of peroxisome proliferator-activated receptor gamma.</a> J Biol Chem. 281, 26943-26950 (2006).</li><li>Tennis, M. A., 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=Sprouty-4+inhibits+transformed+cell+growth,+migration+and+invasion,+and+epithelial-mesenchymal+transition,+and+is+regulated+by+Wnt7A+through+PPARgamma+in+non-small+cell+lung+cancer.">Sprouty-4 inhibits transformed cell growth, migration and invasion, and epithelial-mesenchymal transition, and is regulated by Wnt7A through PPARgamma in non-small cell lung cancer.</a> Mol Cancer Res. 8, 833-843 (2010).</li><li>Steele, J. G., Rowlatt, C., Sandall, J. K., Franks, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+properties+of+high-+and+low-metastatic+cell+lines+selected+from+a+spontaneous+mouse+lung+carcinoma.">Cell surface properties of high- and low-metastatic cell lines selected from a spontaneous mouse lung carcinoma.</a> Int J Cancer. 32, 769-779 (1983).</li></ol>

The authors have nothing to disclose.

A conferma vitro della funzione soppressiva tumorale di proteine ​​di segnalazione è difficile. Uno dei test più rigorosi a disposizione per indagare questa proprietà è il soft agar saggio di formazione di colonie. Qui, abbiamo illustrato il morbido test formazione di colonie agar utilizzando una linea cellulare di carcinoma del polmone murino stabile sovraespressione Wnt7a e Fzd9 rispetto alla sua linea cellulare CMT167 dei genitori. Ci sono diversi punti important...

L&#39;agar morbido dosaggio formazione di colonie è una tecnica largamente usata per valutare trasformazione cellulare in vitro. Storicamente, un kit, la clonogenica, descritto da Puck et al. Nel 1956 è stato utilizzato per valutare la capacità delle cellule di formare colonie 1. In questa tecnica, le cellule sono state disperse su una piastra di coltura e coltivate in presenza di cellule feeder o terreno condizionato per fornire fattori di crescita necessari. Il limite di questa tecnica è che è fornito solo informazioni riguardanti la formazione di colonie. Le cellule normali viene impedito di crescita ancoraggio-indipendente, a causa di un particolare tipo di morte apoptotica, chiamato anoikis 2. Tuttavia, cellule trasformate sono in grado di crescere e dividersi senza legarsi ad un substrato. Per capitalizzare questo concetto, i ricercatori hanno sviluppato il soft agar test formazione di colonie. La morbida agar test formazione di colonie da allora è stato modificato, in anni più recenti, per affrontare spesigenze ecific. Una variante prevede l&#39;incorporazione di colorante fluorometrica per consentire high-throughput conteggio delle colonie. Un&#39;altra variante prevede l&#39;utilizzo di una soluzione di agar specializzata per consentire il recupero di cellule vitali dopo la formazione di colonie quando sono necessari campioni di proteine ​​o di DNA. Nel tradizionale morbido agar saggio formazione di colonie, le cellule sono coltivate in uno strato di agar morbido mescolato con mezzo di coltura cellulare che poggia su un altro strato di agar molle, anche mescolato con mezzo di coltura cellulare, ma che contengono una maggiore concentrazione di agar. Ciò impedisce alle cellule di aderire alla piastra di coltura, ma consente cellule trasformate per formare colonie visibili. La logica dietro questa tecnica è che le cellule normali dipendono da cellula a contatto della matrice extracellulare in grado di crescere e dividere. Al contrario, le cellule trasformate hanno la capacità di crescere e dividere a prescindere dal loro ambiente circostante. Pertanto, cellule capaci di formare colonie in modo di ancoraggio indipendente erano considered trasformare e cancerogene. L&#39;obiettivo generale di questo metodo consiste nel misurare questa capacità nelle cellule in modo semi-quantitativo e rigoroso. La via di segnalazione Wnt è fondamentale in embriogenesi e spesso de-regolato nella tumorigenesi 3-6. Ci sono diversi percorsi associati con segnalazione Wnt. Il percorso canonico prevede di segnalazione Wnt e regolazione della trascrizione genica a valle attraverso i suoi effetti sul coactivator trascrizionale beta-catenina. Wnts segnale anche attraverso diversi percorsi non canonici, per esempio, il percorso planare polarità cellulare, che regola gli elementi coinvolti nella struttura del citoscheletro 7, e la via di Wnt-calcio, che regola il rilascio del calcio dal reticolo endoplasmatico 8. Ligandi Wnt esercitano la loro attività attraverso il legame dei recettori Frizzled. Anche se diversi Wnts hanno dimostrato di essere sovraregolata nel cancro del polmone, Wnt7a ha dimostrato di essere down-regolato in non-smalcancro ai polmoni l cellulare attraverso metilazione del promotore 9. Wnt7a lega Fzd9 e agisce come un soppressore del tumore attraverso un percorso non canonico. Restauro di Wnt-7a e FZD-9 inibisce la crescita di non a piccole cellule di cancro polmonare delle cellule 10. Gli effetti di Wnt7a / Fzd9 sono mediati attraverso l&#39;attivazione di ERK-5, che a sua volta, attiva perossisomi γ del recettore proliferator-activated (PPAR-gamma) 11,12. Qui, abbiamo dimostrato che la sovraespressione di Wnt7a e Fzd9 traduce nella soppressione della crescita ancoraggio-indipendente di una linea cellulare di carcinoma del polmone murino. CMT167 cellule murine sono state derivate da un carcinoma polmonare nei topi C57BL / topi lcrf 13 e sono stati stabilmente trasfettate con Wnt7A e Fzd9. Sovraespressione di Wnt7A e Fzd9 sono stati confermati da-PCR quantitativa (Q-PCR) e la funzionalità di Wnt7A e Fzd9 sovraespressione è stata confermata attraverso l&#39;attivazione a valle del PPAR.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cancer Cell Line of Interest</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">10032302</td>
			<td>CMT-167 Cells</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Powdered RPMI 1640 Medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31800-089</td>
			<td style="width:167px;">Used to prepare 2X cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Liquid RPMI 1650 Medium</td>
			<td style="width:71px;">Cellgro</td>
			<td style="width:119px;">10-040-CV</td>
			<td style="width:167px;">Referred to as 1X cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">HyClone</td>
			<td style="width:119px;">SH30910.03</td>
			<td style="width:167px;">Used to supplement cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">CellGro</td>
			<td style="width:119px;">30-002-Cl</td>
			<td style="width:167px;">Used in cell culture medium.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Difco Noble Agar</td>
			<td style="width:71px;">BD Biosciences</td>
			<td style="width:119px;">214230</td>
			<td style="width:167px;">Used to prepare 1.0% and 0.6% Agar.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP-328-1</td>
			<td style="width:167px;">Used in 2X cell culture medium.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Trypsin</td>
			<td style="width:71px;">Cellgro</td>
			<td style="width:119px;">25-050-Cl</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sterile Bottle-Top Filters</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">09-761-126</td>
			<td style="width:167px;">Used to sterile filter 2X medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Lipofectamine Reagent</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">18324-020</td>
			<td style="width:167px;">Used in PPAR-RE luciferase assay.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">6-well Plates Tissue-culture Treated</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">37 degree C/5% CO2 Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Chemi-Doc Imager</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to take pictures of colonies.&#160;&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Quantity One Software</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to count cell colonies.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15mL Conical Tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">50mL Conical Tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">250mL Erlenmeyer Flasks</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Microwave</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">5mL Serological Pipettes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pipette Aid</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Micropipette</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Hemacytometer w/ cover slip</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pipette Tips</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Inverted Light Microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Centrifuge</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Heat-Resistant Gloves</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Saran Wrap</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice Bucket</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the soft agar colony formation assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

L&#39;espressione di Wnt7A e Fzd9 in CMT167 cellule è efficace nella soppressione del tumore, come illustrato dal nostro morbido agar test formazione di colonie. In via preliminare, abbiamo utilizzato Q-PCR per dimostrare che Wnt7A e Fzd9 mRNA sono espressi in livelli bassi in CMT167 cellule. Cellule CMT167 mostrato bassi livelli di endogena Wnt7A e Fzd9 rispetto a MLE-12 celle, un murino linea cellulare epiteliale polmonare SV40-trasformato (Figura 1). Abbiamo poi trasfettato CMT167 cellule con due ve...

Watch this Scientific Journal Video about The Soft Agar Colony Formation Assay at JoVE.com

The Soft Agar Colony Formation Assay

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Il saggio Formazione morbida Agar Colony

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Research

JoVE Journal

Biology

111.0K Views.  University of Illinois at Chicago. The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Video: The Soft Agar Colony Formation Assay

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Test di adesione ed invasione di Agar in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Ricerca

Biologia

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Supportato bilayers Planar per la formazione degli Studi di sinapsi immunologica e Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Isolare cellule staminali da tessuti molli muscoloscheletrici

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RhoC GTPasi attivazione di analisi

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

Agar-Block Microcosmi per decomposizione controllata tessuti vegetali da Funghi aerobica

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Lievito Colony metodo di inserimento

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenica saggio: cellule aderenti

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Un Matrigel-Based Formazione Assay tubo per valutare l&#39;attività vascolare delle cellule tumorali

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Un dosaggio quantitativo di insulina che esprimono formante colonia Progenitori

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Un approccio semi-quantitativo per valutare la formazione di biofilm mediante lo sviluppo spiegazzato Colony

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...