El trabajo fue apoyado por la financiación básica del Consejo de Investigación Médica. Estamos muy agradecidos a Craig Nicol para preparar los dibujos técnicos. Estamos muy agradecidos a Mateo Pearson y Paul Perry por su apoyo de imágenes.

Todo el trabajo de los animales se realizó de acuerdo con las directrices institucionales bajo licencia por el Ministerio del Interior del Reino Unido (número de licencia del proyecto PPL 60/3785 y 60/4424). 1. Preparación <ol><li> Etiquetar los melanoblastos en la piel en desarrollo mediante la combinación de una línea de ratones que expresan Cre-recombinasa apropiada con una línea de ratones reportero fluorescente. Tyr :: CreA, Tyr :: CREB 8 y Wnt1 :: Cre 14 se han utilizado con éxito como líneas de Cre-expresando y R26YFPR 9 como línea reportero. </li><li> Preparar el medio de cultivo en una campana de flujo laminar; complementar medio Eagle modificado de Dulbecco (DMEM) con (1% v / v de penicilina / estreptomicina), 10% v / v de suero de ternera fetal y 0,584 g / l de L-glutamina. </li><li> El aparato de formación de imágenes en vivo se describe en la Figura 1 esterilizar la base de la cámara de formación de imágenes, al limpiar con etanol al 70%. Esterilizar los insertos, clips de imágenes, y las juntas tóricas remojando overnight en etanol al 70%. Use una tapa estéril de una placa de 6 pocillos que la tapa de la cámara. </li><li> La superficie inferior de la cámara de formación de imágenes se compone de una membrana lummox mantiene en su lugar por una junta tórica (Figura 1). Comience con un gas permeable plato lummox 35 mm. Coloque el plato sobre la cámara de imágenes y cortar la membrana permeable a los gases con una hoja de afeitar afilada sola. Empuje la junta tórica sobre la membrana para asegurar que a la cámara de formación de imágenes. </li><li> Los clips de imágenes (6 en total) sujetan la piel embrionaria en su lugar (Figura 1) antes de su uso, se rellenarán con un 1% de agarosa. Prepare una solución al 2% de agarosa con dH 2 O estéril en el microondas y enfriamiento a 60 ° C. Mezcla 1:1 con 10 ml de medio de cultivo (véase el paso 2) precalentado a 60 ° C. Párese los 6 clips de imagen en una placa de 6 pocillos estériles. Por goteo la solución de agarosa al 1% en el centro de cada clip con una multa Pastette y dejar enfriar. Añadir 1 ml de PBS estéril frío a cada pocillo para evitar la unagarose se sequen. Los clips de formación de imágenes pueden ser almacenadas durante varias horas en PBS. </li><li> Para llevar a cabo la delicada separación de la piel embrionaria y de manejar el tejido diseccionado utilizar el &quot;método del palillo del pelo&quot; como se describe en Kashiwagi et al. 3 En lugar de utilizar un palillo un kebab brocheta de bambú se puede utilizar. Hacer una pequeña hendidura en el extremo plano de la brocheta utilizando una hoja de afeitar afilada única e insertar un cepillo de dientes de cerdas suaves. Recorte el pincho de modo que es de aproximadamente 15 cm de longitud. </li><li> Utilice un microscopio confocal con una cámara ambiental completa o etapa superior de la cámara proporcionando 5% de CO 2 en el aire. Precalentar la platina del microscopio a 37 ° C durante al menos 1 h antes de la formación de imágenes. Esto ayuda a evitar la dispersión de la muestra causado por la expansión de los componentes de la plataforma como se calientan. </li><li> Prepare un conjunto de múltiples posiciones mediante el software de control del microscopio para que se pueda centrar rápida y fácilmente en el medio de cada cultura para hacer una experimentaciónt. </li></ol> 2. Procedimiento <ol><li> Mate de los ratones y designar el primer día post coito como día E0.5. Al día E14.5, sacrificar la hembra por dislocación cervical y exponer la cavidad del cuerpo haciendo una pequeña incisión en la línea media, separando la piel hacia la cabeza y la cola y luego cortar el peritoneo subyacente y repique de vuelta a cada lado del cuerpo. Diseccionar el útero en PBS estéril fría sosteniendo firmemente con unas pinzas y cortar con tijeras a lo largo del mesometrio utilizando tijeras quirúrgicas. Mantenga el útero en hielo hasta su utilización. Para una descripción detallada, véase Shea et al. (2007) 15. </li><li> Diseccionar embriones de útero cortando en primer lugar la longitud de la pared uterina con tijeras quirúrgicas para liberar la decidua y luego la disección de los embriones de la decidua tirando de ellos, aparte de dos pares de pinzas finas. Pantalla para la expresión del indicador fluorescente (si es necesario). </li><li> Cull embriones por decapitación antes de la disección. Subseliminar equently la cola y las extremidades traseras y luego las patas delanteras utilizando una hoja de afeitar afilada sola. Conservar el tejido para el genotipado, si es necesario. </li><li> Hacer una incisión en la zona ventral cerca de la línea media en dirección rostro-caudal. El uso de los palillos del pelo descrito en la etapa 1.6, se burlan cuidadosamente lejos de la piel del tejido conjuntivo subyacente girar el embrión al mismo tiempo. No quite la piel por completo, sino más bien dejar que se adjunta a lo largo del borde más ventral de la primera incisión. </li><li> Es importante no dejar que la piel se arrugó, ya que es entonces muy difícil de montar. Para evitar esto, aplanar la piel usando los palillos del pelo (cara dérmica hacia arriba) en la superficie de un plato de Petri en seco y luego se corta distancia del tronco de embriones utilizando una hoja de afeitar afilada sola. El lado dérmico se puede distinguir de la lado de la epidermis por la observación cuidadosa de la unión entre el tejido diseccionado y el embrión antes de que se retira la piel. El área del tejido diseccionado esaproximadamente 0,5 mm 2 cuando la disección de un embrión E14.5. </li><li> Coloque un clip de formación de imágenes en la parte superior de la piel para que la agarosa está en contacto con el lado dérmico del tejido. Dejar reposar durante 60 segundos y luego, con cuidado se burlan de los bordes del tejido por los lados de la pinza. Invierta el clip y utilizar el pelo se pega a aplanar la piel y eliminar las burbujas de aire, teniendo cuidado de tocar la superficie del tejido (en el área que va a ser fotografiada) lo menos posible. </li><li> Utilice hilo de sutura de seda para atar la piel para el clip de imágenes usando dos nudos simples pulgar a cada lado de la pinza de imágenes para que se contraen y tire de la piel en la ranura cerca de la parte superior del clip. Invierta el clip, empuje interior de la inserción de imágenes y colocar en la base. Llene el inserto con ~ 5 ml de medio de cultivo (véase el paso 1.2). Figura 1 describe el montaje de la cámara de imágenes. </li><li> Repita Protocolo para las 5 muestras restantes. </li></ol> 3. VivirImaging <ol><li> La cámara de imágenes tiene las mismas dimensiones que una placa de 6 pocillos estándar. Utilice un inserto placa apropiada para montar la cámara sobre la platina del microscopio confocal. </li><li> Se requiere una etapa automatizado para la imagen de las culturas en paralelo. Definir las seis posiciones de la imagen en el software confocal. Los ajustes exactos dependerán de la capacidad del microscopio usado. Normalmente, una pequeña z-stack (3-5 z posiciones para dar cuenta de cualquier variación etapa) se utiliza en cada una de las 6 posiciones y una z-stack se captura cada 2 min durante 18 a 24 horas. Para aplicaciones de formación de imágenes en vivo generalmente es ventajoso utilizar una más ancha que ajuste del agujero de alfiler ópticamente óptima para reducir la potencia del láser y el número de z secciones requeridas. </li></ol>

<ol>
	<li>Thomas, A. J., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+making+of+a+melanocyte:+the+specification+of+melanoblasts+from+the+neural+crest.">The making of a melanocyte: the specification of melanoblasts from the neural crest.</a> Pigment Cell &amp; Melanoma Research. 21 (6), 598-610 (2008).</li><li>Mayer, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+migratory+pathway+of+neural+crest+cells+into+the+skin+of+mouse+embryos.">The migratory pathway of neural crest cells into the skin of mouse embryos.</a> Developmental Biology. 34 (1), 39-46 (1973).</li><li>Kashiwagi, M., Huh, N., Turksen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+Culture+of+Developing+Mouse+Skin+and+Its+Application+for+Molecular+Mechanistic+Studies+of+Morphogenesis.">Organ Culture of Developing Mouse Skin and Its Application for Molecular Mechanistic Studies of Morphogenesis.</a> Epidermal Cells: Methods and Protocols. 289, 39-45 (2005).</li><li>Kashiwagi, M., Kuroki, T., Huh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+hair+follicle+formation+by+epidermal+growth+factor+in+an+organ+culture+of+developing+mouse+skin.">Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin.</a> Developmental Biology. 189 (1), 22-32 (1997).</li><li>Van Der Wel, L. I., Wei, L., Prens, E. P., Laman, J. D., Companjen, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+ex+vivo+skin+organ+culture+system+for+functional+studies.">A modified ex vivo skin organ culture system for functional studies.</a> Archives of Dermatological Research. 293 (4), 184-190 (2001).</li><li>Mort, R. L., Hay, L., Jackson, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+live+imaging+of+melanoblast+migration+in+embryonic+mouse+skin.">Ex vivo live imaging of melanoblast migration in embryonic mouse skin.</a> Pigment Cell &amp; Melanoma Research. 23 (2), 299-301 (2010).</li><li>Jordan, S. A. A., Jackson, I. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGF+(KIT+ligand)+is+a+chemokinetic+factor+for+melanoblast+migration+into+hair+follicles.">MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles.</a> Developmental Biology. 225 (2), 424-436 (2000).</li><li>Delmas, V., Martinozzi, S., Bourgeois, Y., Holzenberger, M., Larue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre-mediated+recombination+in+the+skin+melanocyte+lineage.">Cre-mediated recombination in the skin melanocyte lineage.</a> Genesis. 36 (2), 73-80 (2003).</li><li>Srinivas, S., 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=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1 (1), (2001).</li><li>Li, 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=Rac1+Drives+Melanoblast+Organization+during+Mouse+Development+by+Orchestrating+Pseudopod.">Rac1 Drives Melanoblast Organization during Mouse Development by Orchestrating Pseudopod.</a> Driven Motility and Cell-Cycle Progression. 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S., Muccino, D., Rowitch, D. H., Michael, S. K., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+gene+activity+in+mouse+embryos+in+utero+by+a+tamoxifen-inducible+form+of+Cre+recombinase.">Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.</a> Current Biology. 8 (24), 1323-1326 (1998).</li><li>Shea, K., Geijsen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+6.5+dpc+mouse+embryos.">Dissection of 6.5 dpc mouse embryos.</a> Journal of Visualized Experiments. (2), (2007).</li></ol>

Los autores no tienen nada que revelar.

Se describe un método para la cultura de la piel embrionaria que es particularidad susceptible imágenes de células vivas que en microscopios confocal invertido. El método incluye las recientes mejoras que permiten a los 6 cultivos para obtener imágenes en paralelo y elimina la dependencia de matrigel y membranas Nuclepore del método original 6. La diferencia técnica fundamental de técnicas similares es el uso de una membrana permeable a los gases lummox para establecer una interfaz aire-líquido y tam...

Tradicionalmente la piel embrionaria se ha cultivado mediante la disección de la embrión de ratón y de montaje sobre una membrana de policarbonato Nuclepore. La membrana se hace flotar entonces en medio de cultivo, manteniendo así una interfaz aire-líquido a través de la superficie del tejido en desarrollo 3,4. Esta técnica ha sido utilizada para someter a ensayo el comportamiento melanoblasto mediante la fijación del tejido y la evaluación de la distribución melanoblasto utilizando β-galactosidasa como marcador 7. Hemos desarrollado un método que permite melanoblastos de imágenes en directo de la etiqueta fluorescente en ex vivo cultivo de piel 6. A continuación se describe el método en detalle de la disección, para configurar, para vivir de imagen confocal y se incluyen algunas mejoras recientes. Melanoblastos son los precursores embrionarios de los melanocitos, las células que producen el pigmento en el cabello y la piel. Melanoblastos surgen en la cresta neural adyacente al tubo neural en torno al día embrionario 9 (E9) en el ratón en desarrollo embryo. Posteriormente que migran a lo largo de una vía dorsolateral entre el ectodermo y los somitas en desarrollo. En E12.5 se mueven de la dermis a la epidermis, donde proliferan y continúan su migración. El patrón folículo piloso primaria comienza a formarse en la epidermis en E14.5 y E15.5 por melanoblastos están localizando a estos folículos. Para una revisión del desarrollo melanoblasto / melanocito ver Thomas &amp; Erickson (2008) 1. Con el fin de etiquetar la población melanoblasto hemos combinado Tyr :: animales CREB que expresan Cre-recombinasa impulsado por el promotor de la tirosinasa de ratón con 8 animales R26YFPR que expresan la proteína fluorescente amarilla (YFP) condicionalmente desde el locus ROSA26 9. Se describe un método para la piel y la captura de imágenes de cultivo de embriones utilizando un microscopio confocal invertido. Se forma el método original descrito en Mort et al. (2010) 6 está adaptada. La presentemétodo permite formación de imágenes en un formato de 6 pocillos y se elimina la dependencia de las membranas Nuclepore y en Matrigel para apoyar la cultura. En lugar usando un pequeño bloque de agarosa al 1% para estabilizar la piel embrionaria. Extracción de la dependencia de Matrigel es importante, especialmente en situaciones en las que la respuesta de la piel embrionaria a factores de crecimiento solubles es el foco de estudio. Nuestro método original ya se ha utilizado para obtener nuevos conocimientos sobre el desarrollo melanoblasto 10-13 y esperamos que las mejoras que describimos aquí se hacen más potente como una técnica experimental especialmente cuando múltiples culturas paralelas son un requisito.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM high glucose</td>
			<td style="width:132px;">Biochrom AG</td>
			<td style="width:119px;">F0475</td>
			<td style="width:167px;">without phenol red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">P3032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">S9137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum</td>
			<td style="width:132px;">Hyclone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamax</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:119px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Live imaging chamber</td>
			<td style="width:132px;">Custom made</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lummox dishes</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:119px;">94.6077.410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6-well plate</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">657-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single edged razor blade</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">1244-3170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:132px;">Biogene</td>
			<td style="width:119px;">300-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fine pastette</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kebab skewers</td>
			<td style="width:132px;">Waitrose</td>
			<td style="width:119px;"></td>
			<td>Bamboo BBQ skewers 30cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toothbrush</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">633185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Suture thread</td>
			<td style="width:132px;">Look</td>
			<td style="width:119px;">SP115</td>
			<td>Black silk suture thread</td>
		</tr>
	</tbody>
</table>

ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration

Melanoblastos son la cresta neural precursoras de melanocitos derivados; las células responsables de la producción de pigmento en la piel y el cabello. Melanoblastos migran a través de la epidermis de embrión donde posteriormente colonizan el cabello folículos en desarrollo 1,2. Neural migración celular cresta se estudió extensamente in vitro, pero los métodos in vivo todavía no están bien desarrollados, sobre todo en los sistemas de mamíferos. Una alternativa es utilizar ex vivo cultivo organotípico 3-6. Cultura de ratón de la piel embrionaria requiere el mantenimiento de una interfase aire-líquido (ALI) a través de la superficie del tejido 3,6. De alta resolución en vivo de imágenes de ratón de la piel embrionaria se ha visto obstaculizada por la falta de un buen método que no sólo mantiene este ALI sino que también permite la cultura a ser invertida y por lo tanto compatible con distancia de trabajo corta lentes de objetivo y microscopios confocales más. Este artículo describe las mejoras recientes a una metanfetaminadesde que utiliza una membrana permeable a los gases para superar estos problemas y permitir de alta resolución de imagen confocal de la piel embrionaria en cultivo ex vivo 6. Mediante el uso de un melanoblasto Cre-recombinasa específica expresando línea de ratones combinado con la línea reportero R26YFPR podemos etiquetar con fluorescencia la población melanoblasto dentro de estos cultivos de piel. La técnica permite vivir de imágenes de melanoblastos y la observación de su comportamiento y las interacciones con el tejido en el que se desarrollan. Los resultados representativos se incluyen para demostrar la capacidad de vivir-image 6 cultivos en paralelo.

La figura 2 muestra los resultados representativos de un experimento de lapso de tiempo utilizando la piel embrionaria de Tyr :: CREB x embriones R26YFPR en E14.5. 6 cultivos de piel embrionarias fueron imágenes por microscopía confocal cada 2 min durante 18 h. El paquete de software de análisis de imágenes ImageJ software gratuito se utilizó para analizar el comportamiento de los melanoblastos etiquetados-YFP en las 6 películas. Los melanoblastos fueron rastreados automáticamen...

Watch this Scientific Journal Video about Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration at JoVE.com

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Se describe la disección y el cultivo ex vivo de la piel del ratón embrionario. El sistema de cultivo mantiene una interfase aire-líquido a través de la superficie del tejido y permite formación de imágenes en un microscopio invertido. Melanoblastos, un componente de la piel en desarrollo, están marcados con fluorescencia que permite su comportamiento que debe observarse mediante microscopía confocal.

Ex vivo Cultura de embriones de ratón con la piel y en vivo de imágenes de melanoblasto Migración

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

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Biology

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

12.5K vistas.  University of Edinburgh.  Se describe la disección y el cultivo ex vivo de la piel del ratón embrionario. El sistema de cultivo mantiene una interfase aire-líquido a través de la superficie del tejido y permite formación de imágenes en un microscopio invertido. Melanoblastos, un componente de la piel en desarrollo, están marcados con fluorescencia que permite su comportamiento que debe observarse mediante microscopía confocal. 

Video: Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Investigación

Biología

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Imágenes en directo de la migración de células gliales en el ojo del disco imaginal de Drosophila

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Imágenes en vivo del cerebro del embrión de pez cebra mediante microscopía confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Imágenes en directo de Drosophila melanogaster Migraciones hemocitos embrionarias

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo Cultura de células humanas primarias epiteliales del tubo de Falopio

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de la cultura para el corazón de embriones de ratón

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Sistema de Cultura ratón fetal Whole Intestino para<em&gt; Ex Vivo</em&gt; Manipulación de vías de señalización y tridimensional de imágenes en vivo del desarrollo de vellosidades

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

La medición de la rigidez de<em&gt; Ex Vivo</em&gt; Ratón aortas Usando microscopía de fuerza atómica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Methods Article

Ex vivo Cultura de embriones de ratón con la piel y en vivo de imágenes de melanoblasto Migración