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

1. Herstellung von Materialien und Reagenzien <ol><li> Beschriften Sie jede Vertiefung einer Gewebekultur behandelt 6-Well-Platte passend für jede Zelllinie oder der Zustand untersucht. </li><li> Vorbereitung 2x Zellkulturmedium durch Auflösen von 1 g Pulver Medium und 0,2 g Natriumbicarbonat in entionisiertem Wasser auf ein Endvolumen von 50 ml. </li><li> Geben dieses Medium durch ein 0,2-um-Filter sterilisiert. </li><li> Hinzufügen zusätzlicher Komponenten für normale Kultur der Zelllinie von Interesse erforderlich ist. Zum Beispiel wachsen CMT 167 Zelllinie in RPMI 1640-Medium mit 10% FBS und 1% Penicillin / Streptomycin-Lösung ergänzt. Warme Medium auf 37 ° C in heißes Wasserbad vor der Verwendung. </li><li> Bereiten 1x Zellkulturmedium getrennt, wie Sie es für die normale Zellkultur der Zelllinie von Interesse. </li><li> Bereiten Sie 1% Edel Agar durch Zugabe von 1 g edlen Agar auf 100 ml VE-Wasser. HINWEIS: Edel-Agar nicht vollständig unter Rühren alleine lösen. </li><li> Vorbereiten0,6% edle Agar durch Zugabe von 0,6 g Edel Agar in 100 ml entionisiertem Wasser. Beide Lösungen können Agar in 100 ml Glasflaschen mit verschließbarem Deckel für Langzeitlagerung erfolgen. </li><li> Autoklaven wird das edle Agar Mischungen zu sterilisieren. Diese Gemische können im Voraus hergestellt werden und bei 4 ° C gelagert, sondern sollte zum Zeitpunkt des Experiments wieder erhitzt, bis Agar vollständig gelöst werden. </li><li> Vorbereitung Nitroblautetrazoliumchlorid Lösung durch Herstellung einer 1 mg / ml Stammlösung in 1x PBS (8 g NaCl, 0,2 g KCl, 1,44 g Na 2 HPO 4 und 0,24 g KH 2 PO 4 in H 2 O auf ein Endvolumen von 1000 ml ). Dies wird am Ende des Experiments werden die Kolonien zu färben. </li></ol> 2. Überzug der Bodenschicht aus Agar <ol><li> Lösen Sie die Schutzkappe auf der Flasche von 1% Edel Agar und Mikrowelle für ca. 1-2 min. Unter Erhitzen in einer Mikrowelle, überwachen die Lösung eng zu vermeiden Überkochen. Weiter Heizung, beim Mischen mit Unterbrechungen,bis Agar vollständig gelöst und die Lösung klar ist. HINWEIS: Verwenden Sie hitzebeständige Handschuhe, um Kolben nach dem Erhitzen zu behandeln. Geschieht dies nicht, kann Verbrennungen oder schweren Verletzungen führen. </li><li> Zeigen geschmolzen Agarlösung und vorgewärmten 2x Kulturmedium in einem Eiskübel mit heißem Leitungswasser (42 ° C) gefüllt. Legen auch eine 50 ml konischen Röhrchen in einem Rohrhalter im Eiskübel mit heißem Wasser. Übertragen Sie Eimer, um die Zellkultur Kapuze für nachfolgende Schritte. </li><li> Für die untere Schicht aus Agar, benötigen Sie 1,5 ml einer Mischung aus Agar und Medium pro Vertiefung einer 6-Well-Platte. </li><li> Um eine ausreichende Menge der Mischung zu gewährleisten, bereiten insgesamt 12 ml pro 6-Well-Platte. </li><li> Gestartet durch Zugabe von 6 ml Kulturmedium, zu dem 50 ml konischen Röhrchen und dann 6 ml der 1% Edel Agarlösung. </li><li> Kehren Sie die konischen Rohr mehrmals, um zu mischen. Arbeiten bei flottem Tempo wird ein vorzeitiges Aushärten des Soft-Agar zu verhindern. </li><li> Auszuarbeiten etwa 5,5 ml Mischung in eine 5 ml serologische piPette. </li><li> Lassen Sie die Luftblasen nach oben von Pipettensäule vor Hinterlegung 1,5 ml dieser Mischung in jedes Well steigen. Seien Sie vorsichtig, um die Ablagerung von Luftblasen in die Plattenvertiefungen zu vermeiden. </li><li> Die Platten abdecken und lassen Agar Gemisch bei Raumtemperatur verfestigt, in Zellkulturhaube, für 30 min. </li></ol> 3. Überzug der oberen Schicht der Agar enthaltenden Zellen <ol><li> Nachdem die untere Schicht aus Agar sich verfestigt hat, beginnen Herstellung der oberen Schicht. </li><li> Zuerst Ernte die Zellen durch Trypsinierung und verdünnte sie 1: 5 in Kulturmedium (zB 1 ml Trypsin, 4 ml Medium) in eine 15 ml konischen Röhrchen. </li><li> Zählen von Zellen und die Berechnung der Anzahl der benötigten Zellen pro Vertiefung, um eine Zellsuspension zu dieser Zeit vorzubereiten. Diese Zahl wird je nach Zelltyp variieren. Verwenden 5.000 Zellen / Well als Ausgangspunkt und nach Bedarf anpassen. Aus diesem Zellzahl, können Sie eine Zellsuspension von 6667 Zellen / ml vorbereiten würde (dh jeden well werden 0,75 ml dieser Suspension und 0,75 ml des Agar mit einem Gesamtvolumen von 1,5 ml zu erhalten; 2 für eine endgültige Gesamtzellzahl von 5000): die Konzentration der Zellen wird auch 1 verdünnt werden. </li><li> Das Volumen, die pro Vertiefung einer Platte mit 6 Vertiefungen Zellsuspension wird erneut 1,5 ml betragen. Bereiten Sie zusätzliche Zellsuspension in Höhe von 12 ml pro 6-Well-Platte. </li><li> Schmelz 0,6% Agar-Lösung in einer Mikrowelle, wie oben und setzen in Eis Eimer mit heißem Wasser zusammen mit einem 50 ml konischen Röhrchen in einem Rohrhalter und der Zellsuspension aus Schritt 3.3. </li><li> Übertragen Sie die Eiskübel mit geschmolzenem 0,6% Agar, um die Zellkultur Kapuze für nachfolgende Schritte. </li><li> Mischungs 0,6% Agar und Zellsuspension in einem Verhältnis 1: 1, Herstellung eines Gesamtvolumens von 12 ml pro Platte mit 6 Vertiefungen. 1,5 ml pro Vertiefung wird erforderlich sein, aber Extra sollte wie oben durchgeführt werden. </li><li> Pipette 6 ml der Zellsuspension in 50 ml konischen Röhrchen. </li><li> Dann 6 ml 0,6% Agar in das Röhrchen. HINWEIS: Die Temperatur dieser Mischung mussgehalten etwa 42 ° C, um ein vorzeitiges Härten zu vermeiden und das Überleben der Zelle zu maximieren. </li><li> Schnell arbeiten, Pipetten diese Mischung 2-3x um Zellen zu verteilen, dann erarbeiten 5,5 ml Mischung in eine 5 ml serologische Pipette. </li><li> Lassen Sie eventuell vorhandene Luftblasen, um zum Anfang der Pipettensäule vor Hinterlegung 1,5 ml dieser Mischung in jedes Well steigen. Seien Sie vorsichtig, um die Ablagerung von Luftblasen in die Plattenvertiefungen zu vermeiden. </li><li> Erlauben Zelle / Agar Gemisch bei Raumtemperatur, bevor sie in eine 37 ° C befeuchteten Zellkulturbrutschrank zu verfestigen, in Zellkulturhaube, für 30 min. </li><li> Die angemessene Koloniebildung erforderliche Zeit variiert für jede Zelllinie, typischerweise etwa 21 Tage. </li><li> Eine Schicht aus Wachstumsmedium sollte über die obere Agar-Schicht, um ein Austrocknen zu verhindern, aufrechterhalten werden. 100 ul Medium zweimal wöchentlich hinzugefügt ist für diesen Zweck ausreichend. </li></ol> 4. Die Färbung der Platten und das Zählen der Kolonien <ol><li> Flecken Zellen durch Zugabe von 200 _6; l Nitroblau-Tetrazolium-Chlorid-Lösung pro Well über Nacht bei 37 ° C inkubiert Platten. </li><li> Sobald Kolonien gefärbt sind, fotografieren von Brunnen mit einem Imager und zählen Kolonien mit Bildanalyse-Software. </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.

In vitro Bestätigung tumorsuppressive Funktion von Signalproteinen, ist schwierig. Eine der härtesten Tests zur Verfügung, um diese Eigenschaft zu untersuchen ist die Soft-Agar Koloniebildungstest. Hier haben wir die Koloniebildung in Weichagar-Assay unter Verwendung eines Maus-Lungenkarzinom-Zelllinie stabil überexprimieren Wnt7a und FZD9 Vergleich zu seinem Eltern CMT167 Zelllinie dargestellt. Es gibt mehrere wichtige Punkte zu berücksichtigen hinsichtlich der Soft-Agar-Assay...

Die Koloniebildung in Weichagar-Assay ist eine Technik häufig verwendet, um Zelltransformation in vitro zu bewerten. Historisch weiterer Assay, der klonogenen Assay von Puck et al. 1956 wurde verwendet, um die Fähigkeit der Zellen, um Kolonien zu bilden, 1 auszuwerten. In dieser Technik wurden die Zellen auf eine Kulturplatte verteilt und in Gegenwart von &quot;Feeder&quot; Zellen oder konditioniertes Medium die notwendigen Wachstumsfaktoren bereitzustellen gewachsen. Die Einschränkung dieser Technik war, dass es nur zur Verfügung gestellten Informationen über die Koloniebildung. Normale Zellen werden von verankerungsunabhängiges Wachstum gehindert, aufgrund einer bestimmten Art von apoptotischen Tod rief anoikis 2. Jedoch haben transformierten Zellen die Fähigkeit zu wachsen und sich zu teilen, ohne die Bindung an ein Substrat. Um auf diesem Konzept profitieren, entwickelten Forscher die Soft-Agar Koloniebildungstest. Die Koloniebildung in Weichagar-Assay seit modifiziert wurden, in den letzten Jahren zu sp adressierenecific Bedürfnisse. Eine Variante beinhaltet Einbau von fluorimetrischen Farbstoff für Hochdurchsatz-Koloniezählung zu ermöglichen. Eine weitere Variation ist die Verwendung von spezialisierten Agar-Lösung, um abgerufen zu der lebensfähigen Zellen nach der Koloniebildung zu ermöglichen, wenn Protein oder DNA-Proben notwendig. Im herkömmlichen Koloniebildung in Weichagar-Assay werden die Zellen in einer Schicht von weichem Agar mit Zellkulturmedium, das auf einer anderen Schicht aus weichem Agar, auch mit Zellkulturmedium gemischt aufliegt gemischt angebaut, aber mit einer höheren Konzentration von Agar. Dies verhindert, dass Zellen aus dem Kulturplatte haften, noch erlaubt transformierten Zellen zu sichtbaren Kolonien zu bilden. Das Grundprinzip hinter dieser Technik ist, dass normale Zellen abhängen Zelle an extrazelluläre Matrixkontakt in der Lage, zu wachsen und zu verteilen. Umgekehrt transformierten Zellen die Fähigkeit haben, zu wachsen und sich zu teilen, unabhängig von ihrer Umgebung. Daher Zellen in der Lage, Kolonien in einer Verankerung unabhängig bilden waren considered transformiert und krebserregend. Das Gesamtziel dieses Verfahrens ist es, diese Funktion in Zellen in einem semi-quantitative und strenge Weise zu messen. Der Wnt-Signalweg ist entscheidend in der Embryogenese und in der Tumorgenese 3-6 häufig dereguliert. Es gibt verschiedene Wege mit Wnt-Signalweg verbunden. Die kanonische Weg beinhaltet Wnt-Signalisierung und die Regulierung der Transkription stromabwärts über ihre Auswirkungen auf die transkriptionale Koaktivator beta-catenin. Wnts Signal auch durch einige nicht-kanonische Wege, beispielsweise die planare Zellpolarität Weg, die Elemente in Zytoskelettstruktur 7 beteiligt regelt, und der Wnt-Signalweg Calcium, die Freisetzung von Calcium aus dem endoplasmatischen Retikulum 8 regelt. Wnt-Liganden üben ihre Wirkung durch Bindung Frizzled-Rezeptoren. Obwohl mehrere Wnts wurde gezeigt, dass bei Lungenkrebs hochreguliert werden, hat sich gezeigt, Wnt7a in nicht-Smal herunterreguliert werden,l kleinzelligem Lungenkrebs durch Promotormethylierung 9. Wnt7a bindet FZD9 und wirkt als Tumorsuppressor durch eine nicht-kanonische Weg. Wiederherstellung der Wnt-7a und Frizzled-9 hemmt das Wachstum von nicht-kleinzelligen Lungenkrebszellen 10. Die Wirkungen von Wnt7a / FZD9 werden durch die Aktivierung von ERK-5, die wiederum aktiviert Peroxisom-Proliferator-aktivierten Rezeptor γ (PPAR) 11,12 vermittelt. Hier zeigen wir, dass die Überexpression von Wnt7a und FZD9 resultiert in der Unterdrückung des verankerungsunabhängigen Wachstums von einem murinen Lungenkarzinom-Zelllinie. Murine CMT167 Zellen wurden aus einem Lungenkarzinom in C57BL / lcrf Mäusen 13 abgeleitet und wurden stabil mit Wnt7a und FZD9 transfiziert. Eine Überexpression von Wnt7a und FZD9 wurden durch quantitative-PCR (Q-PCR) und der Funktionalität Wnt7a und FZD9 Expression bestätigt wurde durch nachgeschaltete Aktivierung von PPAR &amp; ggr bestätigt.

<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.

Der Ausdruck Wnt7a und FZD9 in CMT167 Zellen wirksam ist Tumorsuppression wie unsere Koloniebildung in Weichagar-Assay veranschaulicht. Vorbereitend, Q-PCR verwendeten wir zu zeigen, dass Wnt7a und FZD9 mRNA sind in geringen Mengen in CMT167 Zellen exprimiert. CMT167 Zellen zeigten geringe endogene Wnt7a und FZD9 Vergleich zu MLE-12-Zellen, eine SV40-transformierte murine Lungen-Epithel-Zelllinie (1). Dann transfiziert CMT167 Zellen mit zwei retroviralen Vektoren, die Überexpression menschlichen Konstr...

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.

Die Soft-Agar Koloniebildungstest

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 Aufrufe.  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.

Serie: 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.

Assay für Adhäsion und 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, ...

Forschung

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.

Unterstützte Planar-Membranen für die Bildung von Study of immunologischen Synapsen und 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.

Isolating Stammzellen aus Soft-Gewebe des Bewegungsapparates

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 GTPase-Aktivierungs-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.  ...

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 Mikrokosmen für Controlled Pflanzengewebekultur Zersetzung durch aerobe Pilze

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.

Hefekolonie Embedding-Methode

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.

Klonogene Test: adhärenter Zellen

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.

Ein Matrigel-Based Rohr Formation Assay zur vaskulären Aktivität von Tumorzellen Beurteilen

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.

A Quantitative Assay für Insulin-exprimierenden Koloniebildende Vorläufern

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.

Ein semi-quantitativer Ansatz, um die Biofilmbildung Mit Wrinkled Colony Entwicklung zu bewerten

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