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

1. Préparation des matériaux et réactifs <ol><li> Étiqueter chaque puits d&#39;une culture de tissu traité plaque de 6 puits appropriée pour chaque ligne ou état cellule objet d&#39;une enquête. </li><li> Préparer 2x milieu de culture cellulaire en dissolvant 1 g de produit de poudre et 0,2 g de bicarbonate de sodium dans de l&#39;eau désionisée à un volume final de 50 ml. </li><li> Passer ce milieu à travers un filtre de 0,2 pm pour stériliser. </li><li> Ajouter des composants supplémentaires nécessaires pour la culture normale de la lignée cellulaire d&#39;intérêt. Par exemple, la ligne 167 CMT croître des cellules dans un milieu RPMI 1640 supplémenté avec 10% de FBS et de la pénicilline / streptomycine solution à 1%. Milieu chaud à 37 ° C dans un bain d&#39;eau chaude avant de les utiliser. </li><li> Préparer 1x milieu de culture cellulaire séparément comme vous le feriez pour la culture cellulaire normale de la lignée cellulaire d&#39;intérêt. </li><li> Préparer la gélose noble à 1% par addition de 1 g de gélose noble à 100 ml d&#39;eau désionisée. REMARQUE: agar Noble ne se dissout pas complètement avec agitation seul. </li><li> Préparer0,6% d&#39;agar noble en ajoutant 0,6 g de gélose noble à 100 ml d&#39;eau désionisée. Les deux solutions d&#39;agar peuvent être réalisées dans des bouteilles de 100 ml en verre avec des couvercles pouvant être fermés pour le stockage à long terme. </li><li> Autoclave les mélanges agar nobles à stériliser. Ces mélanges peuvent être effectués à l&#39;avance et stockées à 4 ° C, mais doivent être chauffées à nouveau au moment de l&#39;expérience jusqu&#39;à dissolution complète de l&#39;agar. </li><li> Préparer une solution de chlorure de tétrazolium nitrobleu en faisant un / ml de solution mère à 1 mg dans 1 x PBS (8 g de NaCl, 0,2 g de KCl, 1,44 g de Na 2 HPO 4 et 0,24 g de KH 2 PO 4 dans H 2 O à un volume final de 1000 ml ). Elle sera utilisée à la fin de l&#39;expérience pour colorer les colonies. </li></ol> 2. Dépôt de couche de fond de Agar <ol><li> Desserrer le bouchon sur la bouteille de 1% d&#39;agar noble et micro-ondes pendant environ 1-2 min. Tout en chauffant dans un four micro-ondes, surveiller étroitement la solution pour éviter de déborder. Continuer à chauffer, tout en mélangeant de façon intermittente,jusqu&#39;à ce que l&#39;agar soit complètement dissous et la solution est claire. REMARQUE: Utiliser des gants résistant à la chaleur pour traiter ballon après le chauffage. Ne pas le faire peut causer des brûlures ou des blessures graves. </li><li> Placez solution d&#39;agar fondu et milieu de culture 2x préchauffé dans un seau à glace rempli avec de l&#39;eau chaude du robinet (42 ° C). Également placer un tube conique de 50 ml dans un support de tube dans le seau à glace avec de l&#39;eau chaude. Transfert seau à la culture cellulaire capot pour les étapes suivantes. </li><li> Pour la couche inférieure de l&#39;agar, vous aurez besoin de 1,5 ml d&#39;un mélange d&#39;agar et milieu par puits d&#39;une plaque à 6 puits. </li><li> Afin d&#39;assurer une quantité suffisante du mélange, préparer un total de 12 ml pour chaque plaque de 6 puits. </li><li> Lancer en ajoutant 6 ml de milieu de culture à 50 ml le tube conique et ensuite 6 ml de la solution de gélose noble à 1%. </li><li> Inverser le tube conique plusieurs fois pour mélanger. Travailler à un rythme soutenu permettra d&#39;éviter un durcissement prématuré de la gélose molle. </li><li> Dresser environ 5,5 ml de mélange dans un 5 ml pi sérologiquepipette. </li><li> Laissez les bulles d&#39;air de remonter en haut de la colonne de la pipette avant de déposer 1,5 ml de ce mélange dans chaque puits. Faites preuve de prudence pour éviter le dépôt de toutes les bulles d&#39;air dans les puits de la plaque. </li><li> Couvrir les plaques et laisser reposer le mélange de la gélose se solidifier à la température ambiante, dans une culture de cellules d&#39;aération, pendant 30 min. </li></ol> 3. Dépôt de la couche supérieure de gélose cellules contenant <ol><li> Une fois que la couche inférieure de la gélose est solidifiée, commencer la préparation de la couche supérieure. </li><li> Tout d&#39;abord, les cellules de récolte par traitement à la trypsine et les diluent 1: 5 dans un milieu de culture (par exemple, pour 1 ml de trypsine, ajouter 4 ml de milieu) dans un tube conique de 15 ml. </li><li> Compter les cellules et calculer le nombre de cellules par puits nécessaires pour préparer une suspension de cellules finale à ce stade. Ce nombre peut varier en fonction du type de cellule. Utilisez 5.000 cellules / puits comme un point de départ et ajuster si nécessaire. Pour ce numéro de cellule, vous préparer une suspension cellulaire de 6667 cellules / ml (soit chaque well recevra 0,75 ml de cette suspension et 0,75 ml de gélose pour un volume total de 1,5 ml; la concentration de cellules est également dilué à 1: 2 pour un nombre total de cellules finale de 5000). </li><li> Le volume de suspension cellulaire nécessaire par puits d&#39;une plaque à 6 puits sera à nouveau 1,5 ml. Préparer supplémentaire suspension cellulaire total de 12 ml par plaque de 6 puits. </li><li> Faire fondre solution de gélose à 0,6% dans un four micro-ondes comme ci-dessus et le placer dans un seau à glace contenant de l&#39;eau chaude avec un tube conique de 50 ml dans un support de tube et la suspension de cellules finale de l&#39;étape 3.3. </li><li> Transférer le seau à glace avec 0,6% de gélose fondue de hotte de culture cellulaire pour les étapes ultérieures. </li><li> Mélanger 0,6% d&#39;agar et de la suspension cellulaire dans un rapport de 1: 1, la préparation d&#39;un volume total de 12 ml par plaque à 6 puits. 1,5 ml seront nécessaires pour bien mais en plus doivent être faites comme ci-dessus. </li><li> Pipette 6 ml de suspension cellulaire dans le tube conique de 50 ml. </li><li> Ensuite, ajouter 6 ml d&#39;agar à 0,6% dans le tube. REMARQUE: La température de ce mélange doit êtremaintenu autour de 42 ° C pour éviter un durcissement prématuré et à maximiser la survie des cellules. </li><li> En travaillant rapidement, pipette ce mélange 2-3x à distribuer des cellules, puis dessinez une hausse de 5,5 ml de mélange dans une pipette de 5 ml sérologique. </li><li> Autoriser les bulles d&#39;air de remonter en haut de la colonne de la pipette avant de déposer 1,5 ml de ce mélange dans chaque puits. Faites preuve de prudence pour éviter le dépôt de toutes les bulles d&#39;air dans les puits de la plaque. </li><li> Laisser le mélange de cellules / gélose se solidifier à la température ambiante, dans une culture de cellules d&#39;aération, pendant 30 minutes avant de les placer dans un 37 ° C culture cellulaire incubateur humidifié. </li><li> Le temps nécessaire à la formation de colonies adéquate varie pour chaque lignée cellulaire, typiquement environ 21 jours. </li><li> Une couche de milieu de croissance doit être maintenue au-dessus de la couche supérieure de gélose pour éviter la dessiccation. 100 pi de milieu ajoutée deux fois par semaine est suffisant à cet effet. </li></ol> 4. la coloration des plaques et comptage des colonies <ol><li> cellules de tache en ajoutant 200 _6, l d&#39;une solution de chlorure de nitrobleu de tétrazolium par puits et en incubant les plaques pendant une nuit à 37 ° C. </li><li> Une fois que les colonies sont colorées, prendre des photographies des puits à l&#39;aide d&#39;un imageur et compter les colonies en utilisant un logiciel d&#39;analyse d&#39;images. </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. 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The authors have nothing to disclose.

En confirmation in vitro de la fonction de suppresseur de tumeur de protéines de signalisation est difficile. L&#39;un des tests les plus rigoureux disponibles pour enquêter sur cette propriété est le dosage de formation de colonies sur gélose molle. Ici, nous avons représenté le dosage de formation de colonies sur gélose molle en utilisant une lignée cellulaire de carcinome du poumon de souris surexprimant de manière stable et Wnt7a Fzd9 par rapport à sa lignée cellulaire parentale CMT167....

Le dosage de formation de colonies sur gélose molle est une technique largement utilisée pour évaluer la transformation cellulaire in vitro. Historiquement, un autre test, le test clonogénique, décrit par Puck et al., En 1956, a été utilisé pour évaluer la capacité des cellules à former des colonies 1. Dans cette technique, les cellules ont été dispersées sur une plaque de culture et cultivées en présence de cellules nourricières »ou« milieu conditionné pour fournir des facteurs de croissance nécessaires. La limitation de cette technique est qu&#39;elle ne fourni des informations concernant la formation de colonies. Les cellules normales sont empêchés de croissance indépendante de l&#39;ancrage, en raison d&#39;un type particulier de la mort par apoptose, appelé anoïkis 2. Toutefois, les cellules transformées ont la capacité de croître et de se diviser sans se lier à un substrat. Pour capitaliser sur ce concept, les chercheurs ont développé le test de formation de colonies sur gélose molle. Le test de formation de colonies sur gélose molle a depuis été modifié, au cours des dernières années, pour répondre specific besoins. Une variante consiste incorporation de colorant fluorimétrique pour permettre à haut débit comptage de colonies. Une autre variante consiste à utiliser une solution d&#39;agar spécialisé pour permettre la récupération de cellules viables après la formation de colonies lorsque les échantillons de protéines ou d&#39;ADN sont nécessaires. Dans le dosage souple classique de formation de colonies agar, les cellules sont cultivées dans une couche d&#39;agar mou mélangé avec un milieu de culture cellulaire qui repose sur une couche de gélose molle, également mélangée avec du milieu de culture de cellules, mais contenant une concentration plus élevée de la gélose. Cela empêche les cellules d&#39;adhérer à la plaque de culture, tout en permettant aux cellules transformées de former des colonies visibles. La raison de cette technique est que les cellules normales de la cellule dépendent de la matrice extracellulaire de contact pour être en mesure de croître et de se diviser. A l&#39;inverse, les cellules transformées sont capables de croître et de se diviser sans tenir compte de leur environnement. Par conséquent, des cellules capables de former des colonies d&#39;une manière indépendante de l&#39;ancrage sont considered à transformer et cancérigènes. L&#39;objectif global de cette méthode est de mesurer cette capacité dans les cellules d&#39;une manière semi-quantitative et rigoureuse. La voie de signalisation Wnt est critique dans l&#39;embryogenèse et souvent dérégulé dans la tumorigenèse 6.3. Il existe de multiples voies impliquant la signalisation Wnt. La voie canonique implique la signalisation Wnt et régulation de la transcription du gène en aval par ses effets sur la co-activateur transcriptionnel beta-caténine. Le signal Wnt également à travers plusieurs voies non canoniques, par exemple, la voie plane de la polarité cellulaire, qui régule des éléments participant à la structure du cytosquelette 7, et la voie Wnt-calcium, qui régule la libération de calcium à partir du reticulum endoplasmique 8. Ligands Wnt exercent leur activité grâce à des récepteurs Frizzled contraignant. Bien que plusieurs Wnt ont été montré pour être régulés à la hausse dans le cancer du poumon, Wnt7a a été montré pour être régulé à la baisse en non-Smalle cancer du poumon de cellules L à travers la méthylation du promoteur 9. Wnt7a lie Fzd9 et agit comme un suppresseur de tumeur par l&#39;intermédiaire d&#39;une voie non-canonique. La restauration de Wnt-7a et FZD-9 inhibe la croissance de non-petites cellules du cancer du poumon à grandes cellules 10. Les effets de Wnt7a / Fzd9 sont médiés par l&#39;activation de ERK-5, ce qui, à son tour, active peroxisome γ du récepteur activé par les proliférateurs de (PPARy) 11,12. Ici, nous montrons que la surexpression de Wnt7a Fzd9 et se traduit par la suppression de la croissance indépendante de l&#39;ancrage d&#39;une lignée de cellules de carcinome pulmonaire murin. Cellules CMT167 murins ont été dérivées d&#39;un carcinome du poumon chez les souris C57BL / souris LCRF 13 et ont été transfectées de manière stable avec Wnt7a et Fzd9. La surexpression de Wnt7a Fzd9 et ont été confirmés par PCR quantitative (Q-PCR) et la fonctionnalité de Wnt7a et Fzd9 surexpression a été confirmé par l&#39;activation en aval de PPARy.

<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>
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		</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;expression de Wnt7a et Fzd9 dans CMT167 cellules est efficace dans la suppression des tumeurs, comme illustré par notre essai de formation de colonies sur gélose molle. Préalablement, nous avons utilisé Q-PCR pour montrer que WNT7A et Fzd9 ARNm sont exprimés dans de faibles niveaux dans CMT167 cellules. CMT167 cellules ont montré de faibles niveaux de Wnt7a endogène et Fzd9 par rapport aux cellules MLE-12, une lignée de cellules épithéliales pulmonaires murines transformées par SV40 (figure 1).<...

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.

La formation de dosage souple 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

110.8K vues.  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.

Vidéo: 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.

Dosage de l&#39;adhésion et l&#39;invasion Agar chez S. cerevisiae

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

Recherche

Biologie

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.

Soutenu bicouches planaires pour la formation des synapses immunologiques Etude de et 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.

Isoler des cellules souches à partir des tissus mous musculo-squelettiques

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

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 microcosmes pour la décomposition des tissus végétaux contrôlés par des champignons aérobie

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.

Méthode d&#39;insertion de levure Colony

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.

Test clonogénique: cellules adhérentes

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 test basé sur Matrigel Tube Formation d&#39;évaluer l&#39;activité Vasculogenic de cellules tumorales

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 dosage quantitatif de l&#39;insuline exprimer formant colonie Progéniteurs

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.

Une approche semi-quantitative pour évaluer la formation de biofilms utilisant le développement des colonies ridée

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

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Des cellules endothéliales Tube Formation test pour la<em&gt; In Vitro</em&gt; Etude de l&#39;angiogenèse

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...