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

1. Preparación de Materiales y Reactivos <ol><li> Etiquetar cada pocillo de un cultivo de tejido tratado placa de 6 pocillos adecuada para cada línea celular o condición objeto de la investigación. </li><li> Preparar medio de cultivo celular 2x disolviendo 1 g de medio en polvo y 0,2 g de bicarbonato de sodio en agua desionizada hasta un volumen final de 50 ml. </li><li> Pasar este medio a través de un filtro de 0,2 micras para esterilizar. </li><li> Añadir componentes adicionales necesarios para el cultivo normal de la línea celular de interés. Por ejemplo, crecer CMT línea 167 de células en medio RPMI 1640 suplementado con 10% de FBS y 1% de penicilina / estreptomicina solución. Medio caliente a 37 ° C en baño de agua caliente antes de su uso. </li><li> Preparar medio de cultivo celular 1x por separado como si se tratara de la cultura normal de las células de la línea celular de interés. </li><li> Preparar 1% de agar noble mediante la adición de 1 g de agar noble a 100 ml de agua desionizada. NOTA: agar Noble no se disolverá por completo con agitación por sí sola. </li><li> Preparar0,6% de agar noble mediante la adición de 0,6 g de agar noble a 100 ml de agua desionizada. Ambas soluciones de agar se pueden hacer en botellas de 100 ml de vidrio con tapas de cierre, para su almacenamiento a largo plazo. </li><li> Autoclave las mezclas de agar nobles para esterilizar. Estas mezclas se pueden hacer de antemano y se almacenan a 4 ° C, pero deben ser calentados de nuevo en el momento del experimento hasta el agar se haya disuelto completamente. </li><li> Preparar una solución de cloruro de tetrazolio nitroazul haciendo una solución madre de 1 mg / ml en 1X PBS (8 g NaCl, 0,2 g KCl, 1,44 g de Na 2 HPO 4, y 0,24 g KH 2 PO 4 en H 2 O hasta un volumen final de 1000 ml ). Esto se utiliza al final del experimento para teñir las colonias. </li></ol> 2. Revestimiento del de la capa inferior de Agar <ol><li> Afloje el tapón de la botella de 1% de agar noble y microondas durante unos 1-2 minutos. Mientras que el calentamiento en un microondas, un seguimiento de la solución cuidadosamente para evitar desborde. Continuar calentando, mientras se mezcla de forma intermitente,agar hasta que se haya disuelto completamente y la solución es clara. NOTA: Utilice guantes resistentes al calor para manejar frasco después del calentamiento. Si no lo hace puede provocar quemaduras o lesiones graves. </li><li> Coloque solución de agar fundido y medio de cultivo 2x pre-calentado en un cubo de hielo llena de agua caliente de la llave (42 ° C). También colocar un tubo cónico de 50 ml en un soporte de tubo en el cubo de hielo con agua caliente. Traslado cubo para campana de cultivo de células para las etapas subsiguientes. </li><li> Para la capa inferior de agar, necesitará 1,5 ml de una mezcla de agar y medio por pocillo de una placa de 6 pocillos. </li><li> Para asegurar una cantidad adecuada de la mezcla, preparar un total de 12 ml para cada placa de 6 pocillos. </li><li> Iniciar mediante la adición de 6 ml de medio de cultivo al tubo cónico de 50 ml y luego 6 ml de la solución de agar noble 1%. </li><li> Invierta el tubo cónico varias veces para mezclar. Trabajando a un ritmo acelerado impedirá el endurecimiento prematuro del agar blando. </li><li> Elaborar aproximadamente 5,5 ml de la mezcla en un 5 ml pi serológicapette. </li><li> Permitir que las burbujas de aire a la altura de la parte superior de la columna de pipeta antes de depositar 1,5 ml de esta mezcla en cada pocillo. Tenga cuidado para evitar la deposición de las burbujas de aire en los pocillos de la placa. </li><li> Cubrir las placas de agar y permitir que la mezcla se solidifique a temperatura ambiente, en campana de cultivo celular, durante 30 min. </li></ol> 3. Chapado de la capa superior de células que contienen Agar <ol><li> Una vez que la capa inferior de agar se ha solidificado, comenzar la preparación de la capa superior. </li><li> Primero, las células de la cosecha mediante tripsinización y se les diluyen 1: 5 en medio de cultivo (por ejemplo, para 1 ml de tripsina, añadir 4 ml de medio) en un tubo cónico de 15 ml. </li><li> Contar las células y calcular el número de células por pocillo necesarios para preparar una suspensión celular final en este momento. Este número variará dependiendo del tipo de célula. Utilice 5.000 células / así como un punto de partida y ajustar según sea necesario. Para este número de células, que le prepare una suspensión celular de 6.667 células / ml (es decir, cada well recibirá 0,75 ml de esta suspensión y 0,75 ml de agar para un volumen total de 1,5 ml; la concentración de células también se diluyó 1: 2 para un recuento final de células total de 5000). </li><li> El volumen de la suspensión celular necesaria por pocillo de una placa de 6 pocillos será de nuevo 1,5 ml. Preparar suspensión celular adicional por un total de 12 ml por placa de 6 pocillos. </li><li> Derretir solución de agar 0,6% en un horno de microondas como anteriormente y colocar en el cubo de hielo que contiene agua caliente junto con un tubo cónico de 50 ml en un soporte de tubo y la suspensión celular final de la Etapa 3.3. </li><li> Transferir el cubo de hielo derretido con 0,6% de agar para campana de cultivo de células para las etapas subsiguientes. </li><li> Mezclar 0,6% de agar y la suspensión celular en una relación 1: 1, la preparación de un volumen total de 12 ml por placa de 6 pocillos. Se requerirá 1,5 ml por pocillo, pero adicional deben hacerse como anteriormente. </li><li> Pipetear 6 ml de la suspensión celular en el tubo cónico de 50 ml. </li><li> A continuación, añadir 6 ml de agar al 0,6% al tubo. NOTA: La temperatura de esta mezcla debe sermantenido en torno a los 42 ° C para evitar el endurecimiento prematuro y para maximizar la supervivencia celular. </li><li> Trabajando rápidamente, pipeta esta mezcla 2-3x para distribuir las células, a continuación, dibuje un 5,5 ml de la mezcla en un 5 ml pipeta serológica. </li><li> Permita que las burbujas de aire suban a la parte superior de la columna de la pipeta antes de depositar 1,5 ml de esta mezcla en cada pocillo. Tenga cuidado para evitar la deposición de las burbujas de aire en los pocillos de la placa. </li><li> Deje que la mezcla de células / agar para solidificar a temperatura ambiente, en campana de cultivo celular, durante 30 min antes de colocar en un 37 ° C humidificado incubadora de cultivo celular. </li><li> El tiempo requerido para la formación de colonias adecuada varía para cada línea celular, típicamente alrededor de 21 días. </li><li> Una capa de medio de crecimiento debe mantenerse sobre la capa superior de agar para evitar la desecación. 100 l de medio añadido dos veces por semana es suficiente para este propósito. </li></ol> 4. tinción de las placas y contando Colonias <ol><li> Las células de la mancha mediante la adición de 200 _6; l de solución de cloruro de tetrazolio nitroazul por pocillo e incubando las placas durante la noche a 37 ° C. </li><li> Una vez que las colonias se tiñen, tome fotografías de pozos utilizando un generador de imágenes y cuente las colonias utilizando el software de análisis de imágenes. </li></ol>

<ol>
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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. 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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 confirmación vitro de la función supresora de tumor de las proteínas de señalización es difícil. Uno de los ensayos más rigurosos disponibles para investigar esta propiedad es el ensayo de formación de colonias en agar blando. Aquí, hemos ilustrado el ensayo de formación de colonias en agar suave utilizando una línea celular de carcinoma de pulmón murino de forma estable y que sobreexpresan Wnt7a Fzd9 comparación con su padres línea celular CMT167. Hay vari...

El ensayo de formación de colonias en agar blando es una técnica ampliamente utilizada para evaluar la transformación celular in vitro. Históricamente, se utilizó otro ensayo, el ensayo clonogénico, descrito por Puck et al. En 1956 para evaluar la capacidad de las células para formar colonias 1. En esta técnica, las células se dispersaron en una placa de cultivo y se cultivaron en la presencia de células alimentadoras &#39;&#39; o medio acondicionado para proporcionar factores de crecimiento necesarios. La limitación de esta técnica es que sólo se proporciona información con respecto a la formación de colonias. Las células normales se impide el crecimiento independiente de anclaje, debido a un tipo particular de muerte apoptótica, denominado anoikis 2. Sin embargo, las células transformadas tienen la capacidad de crecer y dividirse sin unirse a un sustrato. Para sacar provecho de este concepto, los investigadores desarrollaron el ensayo de formación de colonias en agar blando. El ensayo de formación de colonias en agar blando desde entonces ha sido modificada, en años más recientes, para abordar specific necesidades. Una variación consiste en la incorporación de colorante fluorométrico para permitir de alto rendimiento recuento de colonias. Otra variación implica el uso de solución especializada agar para permitir la recuperación de células viables después de la formación de colonias cuando se necesitan muestras de proteínas o de ADN. En el ensayo de formación de colonias en agar blando tradicional, las células se cultivan en una capa de agar blando mezclado con medio de cultivo celular que descansa sobre otra capa de agar blando, también mezclado con medio de cultivo celular, pero que contiene una mayor concentración de agar. Esto evita que las células se adhieran a la placa de cultivo, sin embargo, permite que las células transformadas para formar colonias visibles. El fundamento de esta técnica es que las células normales dependen de contacto célula a la matriz extracelular para ser capaz de crecer y dividirse. Por el contrario, las células transformadas tienen la capacidad de crecer y dividirse independientemente de su entorno. Por lo tanto, las células capaces de formar colonias de una manera independiente de anclaje fueron considered a transformar y cancerígeno. El objetivo general de este método es medir esta capacidad en las células de una manera semi-cuantitativa y rigurosa. La vía de señalización Wnt es esencial en la embriogénesis y, a menudo reguladas des en la tumorigénesis 3-6. Hay múltiples vías asociadas con la señalización de Wnt. La vía canónica implica la señalización y la regulación de la transcripción de genes Wnt aguas abajo a través de sus efectos sobre el coactivador transcripcional de beta-catenina. Wnts también la señal a través de varias vías no canónicos, por ejemplo, la vía de la polaridad celular planar, que regula los elementos involucrados en la estructura del citoesqueleto 7, y de la vía Wnt-calcio, que regula la liberación de calcio desde el retículo endoplásmico 8. Ligandos Wnt ejercen su actividad a través de unirse a receptores Frizzled. Aunque varios Wnt han demostrado ser upregulated en el cáncer de pulmón, Wnt7a ha demostrado ser regulada en el no-Smalcáncer de pulmón l de células a través de la metilación del promotor 9. Wnt7a une Fzd9 y actúa como un supresor de tumor a través de una vía no canónica. Restauración de Wnt-7a y Fzd-9 inhibe el crecimiento de células no pequeñas de cáncer de pulmón de células 10. Los efectos de la Wnt7a / Fzd9 están mediadas a través de la activación de ERK-5, que a su vez, activa los receptores γ proliferador de peroxisoma activados (PPAR) 11,12. Aquí, mostramos que la sobreexpresión de Wnt7a y Fzd9 se traduce en la supresión del crecimiento independiente de anclaje de una línea celular de carcinoma de pulmón murino. CMT167 células murinas se derivaron de un carcinoma de pulmón en ratones C57BL / LCRF 13 y fueron transfectadas establemente con Wnt7a y Fzd9. La sobreexpresión de Wnt7a y Fzd9 fueron confirmados por PCR cuantitativa (Q-PCR) y la funcionalidad de Wnt7a y Fzd9 sobreexpresión fue confirmada a través de la activación de aguas abajo de PPAR.

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

La expresión de Wnt7a y Fzd9 en CMT167 células es eficaz en la supresión de tumores, como se ilustra por nuestro ensayo de formación de colonias en agar blando. Preliminarmente, se utilizó Q-PCR para demostrar que Wnt7a y Fzd9 ARNm se expresan en niveles bajos en CMT167 células. CMT167 células mostraron bajos niveles de endógeno Wnt7a y Fzd9 cuando se compara con MLE-12 células, un murino línea de células epiteliales de pulmón transformada con SV40 (Figura 1). A continuación, las células t...

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.

El ensayo de formación de colonias en agar blando

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

110.6K vistas.  University of Illinois at Chicago. The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Video: The Soft Agar Colony Formation Assay

Assay for Adhesion and Agar Invasion in S. cerevisiae

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

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

Ensayo de adhesión y la invasión de agar en S. cerevisiae

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

Investigación

Biología

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.

El apoyo de bicapa plana para la Formación de Estudio de la sinapsis inmunológica y 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.

Aislamiento de células madre de tejidos blandos musculoesqueléticos

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 GTPasa ensayo de activación

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

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.

Colonia de levaduras Método de anidamiento

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.

Ensayo clonigénicas: las células adherentes

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.

A Matrigel basado en tubo de ensayo de formación para evaluar la actividad de las células tumorales vasculogénica

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 análisis cuantitativo de la insulina que expresan la formación de colonias de progenitores

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

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

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

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

Un enfoque semi-cuantitativo para evaluar la formación de biopelículas Con el desarrollo de colonias arrugada

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.

Ensayo de formación de tubos de células endoteliales para la<em&gt; In Vitro</em&gt; Estudio de la angiogénesis

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