O trabalho foi apoiado pelo financiamento de base do Conselho de Investigação Médica. Somos gratos a Craig Nicol para preparar desenhos técnicos. Somos gratos a Matthew Pearson e Paul Perry por seu apoio de imagem.

Todo o trabalho animal foi executada de acordo com as diretrizes institucionais sob licença pela UK Home Office (número de licença do projeto PPL 60/3785 e 60/4424). 1. Preparação <ol><li> Identifique os melanoblastos na pele em desenvolvimento, combinando uma linha de expressar rato Cre-recombinase apropriado com uma linha de rato repórter fluorescente. :: CreA Tyr, Tyr :: CREB 8 e Wnt1 :: Cre 14 têm sido utilizados com sucesso como linhas de Cre-expressando e R26YFPR 9 como uma linha de repórter. </li><li> Preparar o meio de cultura numa câmara de fluxo laminar; complementar de Dulbecco Modified Eagle Médium (DMEM) com 1% (v / v de Penicilina / Estreptomicina), 10% v / v de soro fetal de vitelo e 0,584 g / L de L-glutamina. </li><li> O aparelho de imagem ao vivo está descrito na Figura 1 Esterilizar a base da câmara de imagiologia, limpando-se com etanol a 70%. Esterilizar as inserções, clipes de imagem, e O-rings por imersão overnight em etanol a 70%. Use uma tampa estéril a partir de uma placa de 6 poços, como a tampa da câmara. </li><li> A superfície do fundo da câmara de imagem é composto por uma membrana lummox mantido no lugar por um anel em O (Figura 1). Comece com um 35 milímetros gás permeável lummox prato. Coloque o prato sobre a câmara de imagem e cortar a membrana permeável a gás com uma única lâmina de barbear gumes. Empurrar o anel de vedação ao longo da membrana para assegurar que a câmara de imagem. </li><li> Os clipes de imagem (6 no total) fixar a pele embrionária no local (Figura 1) antes do uso, devem ser preenchidos com 1% de agarose. Prepara-se uma solução de 2% de agarose com dH 2 O estéril por microondas e arrefecida a 60 ° C. Mistura a 1:1 com 10 ml de meio de cultura (ver passo 2) pré-aquecido a 60 ° C. Levante os seis clipes de imagens em um estéril 6 bem-placa. Escorrer a solução de agarose a 1% para o centro de cada grampo usando um fino pastette e deixa-se arrefecer. Adicionar 1 ml de PBS estéril frio a cada poço para evitar a umgarose sequem. Os clipes de imagens podem ser armazenadas durante várias horas em PBS. </li><li> Para levar a cabo a separação de pele delicada embrionário e de manipular o tecido dissecado utilizar o &#39;método vara cabelo &quot;, tal como descrito em Kashiwagi et al. 3 Em vez de usar um pauzinho um kebab espeto de bambu podem ser usadas. Faça um pequeno corte na extremidade plana do espeto usando uma única lâmina de barbear afiada e inserir uma escova de dentes de cerdas macias. Apare o espeto de modo que é de aproximadamente 15 cm de comprimento. </li><li> Usar um microscópio confocal com uma câmara ambiental completo ou fase superior da câmara fornecer 5% de CO 2 no ar. Pré-aquecer o estágio de microscópio de 37 ° C durante pelo menos 1 hora antes da imagiologia. Isso ajuda a impedir a dispersão da amostra causada pela expansão dos componentes do estágio como eles se aquecer. </li><li> Preparar um conjunto multi-posição usando o software de controle do microscópio para que se possa rapidamente e facilmente centralizar no meio de cada cultura para criar uma experimentaçãot. </li></ol> 2. Procedimento <ol><li> Companheiro os ratos e designar o primeiro dia pós coitum como o dia E0.5. No dia E14.5, abater a fêmea por deslocamento cervical e expor a cavidade do corpo através de uma pequena incisão na linha média, separando a pele na direcção da cabeça e cauda e, em seguida, o corte do peritoneu subjacente e repicante volta para qualquer um dos lados do corpo. Dissecar o útero em frio PBS estéril, segurando firmemente com uma pinça e cortando ao longo da mesométrio usando uma tesoura cirúrgica. Manter o útero no gelo até ser necessária. Para uma descrição detalhada ver Shea et al. (2007) 15. </li><li> Dissecar de embriões no útero, em primeiro lugar o corte do comprimento da parede uterina com tesouras cirúrgicas para libertar a decídua e dissecando os embriões do decidua por separá-las com os dois pares de pinças finos. Tela para a expressão repórter fluorescente (se necessário). </li><li> Cull embriões por decapitação antes da dissecção. Subsequently retirar a cauda e membros posteriores e depois os membros anteriores usando uma única lâmina de barbear gumes. Reter tecido para genotipagem, se necessário. </li><li> Faça uma incisão na ventrum perto da linha média em uma direção rostro-caudal. Usando as varas do cabelo descritos na etapa 1.6, cuidadosamente provocar longe da pele a partir do tecido conjuntivo subjacente rodar o embrião ao mesmo tempo. Não remova a pele completamente, mas sim deixá-lo ligado ao longo da borda mais distante ventral da primeira incisão. </li><li> É importante não deixar que a pele se tornar asneira, pois é então muito difícil de montar. Para evitar isso, alisar a pele usando os paus de cabelo (lado dérmico para cima) sobre a superfície de uma placa de Petri, seco e, em seguida, cortado a partir do tronco de embriões utilizando uma única lâmina de barbear afiada. O lado dérmico pode ser distinguido do lado epidérmico por observação cuidadosa da junção entre o tecido dissecado e o embrião, antes que a pele é retirada. A área de tecido é dissecadocerca de 0,5 mm 2 quando dissecando o embrião E14.5. </li><li> Coloque um grampo de imagem em cima da pele, de modo que a agarose é tocar o lado dérmico do tecido. Deixar em repouso durante 60 segundos e, em seguida, cuidadosamente provocar as bordas do tecido acima dos lados do clipe. Inverter o grampo e usar o cabelo varas para alisar a pele e remover quaisquer bolhas de ar, tendo o cuidado de tocar a superfície do tecido (na zona que vai ser trabalhada) tão pouco quanto possível. </li><li> Use fio de sutura de seda para amarrar a pele para o clipe de imagiologia utilizando dois nós do polegar simples em qualquer um dos lados do clipe de imagem de modo que apertar e retirar a pele para dentro da ranhura estreita para a parte superior do grampo. Inverta o clipe, empurre para dentro a inserção de imagens e coloque na base. Encha a inserção com ~ 5 ml de meio de cultura (veja o passo 1.2). Figura 1 apresenta a montagem da câmara de imagem. </li><li> Repita Protocolo para os restantes 5 amostras. </li></ol> 3. ViverImagem <ol><li> A câmara de imagem tem as mesmas dimensões que uma placa de 6 poços padrão. Use uma placa de inserção apropriado para montar a câmara na fase do microscópio confocal. </li><li> Uma fase automatizada é necessária para a imagem das culturas em paralelo. Definir as seis posições de imagem no software confocal. As definições exactas irão depender da capacidade do microscópio utilizado. Tipicamente, uma pequena pilha z-(3-5 z-posições para ter em conta qualquer variação fase) é usado em cada uma das seis posições e uma pilha z é capturado a cada 2 min durante 18-24 h. Para aplicações de live-imagem geralmente é vantajoso usar uma definição mais ampla do que pinhole opticamente ideal para reduzir a potência do laser e do número de z-seções necessárias. </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. Developmental Cell. 21 (4), 722-734 (2011).</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=Activated+Mutant+NRas(Q61K)+Drives+Aberrant+Melanocyte+Signaling,+Survival,+and+Invasiveness+via+a+Rac1-Dependent+Mechanism.">Activated Mutant NRas(Q61K) Drives Aberrant Melanocyte Signaling, Survival, and Invasiveness via a Rac1-Dependent Mechanism.</a> The Journal of Investigative Dermatology. , 1-12 (2012).</li><li>Schachtner, H., 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=Tissue+inducible+Lifeact+expression+allows+visualization+of+actin+dynamics+in+vivo+and+ex+vivo.">Tissue inducible Lifeact expression allows visualization of actin dynamics in vivo and ex vivo.</a> European Journal of Cell Biology. 91 (11-12), 923-929 (2012).</li><li>Ma, Y., 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=Fascin+1+is+transiently+expressed+in+mouse+melanoblasts+during+development+and+promotes+migration+and+proliferation.">Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation.</a> Development. 140 (10), 2203-2211 (2013).</li><li>Danielian, P. 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>

Os autores não têm nada a revelar.

Nós descrevemos um método para a cultura da pele embrionária que é particularidade passíveis de imagem de células vivas para a microscopia confocal invertidos. O método inclui os melhoramentos recentes que permitem seis culturas a ser trabalhada em paralelo e elimina a dependência de matrigel e membranas Nuclepore do método original 6. A diferença fundamental técnica a partir de técnicas similares é a utilização de uma membrana permeável ao gás lummox para estabelecer uma interface líquido d...

Tradicionalmente pele embrionária foi cultivada por dissecar a partir do embrião do rato e montagem em uma membrana de policarbonato Nuclepore. A membrana é então lançada em meio de cultura, mantendo, assim, uma interface ar-líquido através da superfície do tecido em desenvolvimento 3,4. Esta técnica tem sido utilizada para avaliar o comportamento melanoblasto pela fixação do tecido e a avaliação de distribuição melanoblasto usando β-galactosidase como marcador 7. Nós desenvolvemos um método que permite melanoblastos live-imaging de fluorescente etiquetado em ex vivo cultura pele 6. Aqui vamos descrever o método em detalhes de dissecação, a configuração, a viver de imagem confocal e incluem algumas melhorias recentes. Melanoblastos são os precursores embrionários dos melanócitos, as células que produzem o pigmento no cabelo e na pele. Melanoblastos surgem na crista neural adjacente ao tubo neural embrionária por volta do dia 9 (E9) no rato em desenvolvimento embryo. Subsequentemente migram ao longo de um percurso entre a dorsolateral ectoderme e somitos desenvolvimento. No E12.5 eles se movem da derme para a epiderme, onde proliferam e continuar sua migração. O padrão primário folículo piloso começa a se formar na epiderme em E14.5 e E15.5 por melanoblastos está localizando a esses folículos. Para uma revisão do desenvolvimento melanoblasto / melanócito ver Thomas &amp; Erickson (2008) 1. A fim de marcar a população melanoblasto combinamos Tyr :: CREB animais que expressam Cre-recombinase, conduzido pelo promotor de rato de tirosinase com 8 animais R26YFPR que expressam a proteína fluorescente amarela (YFP) condicionalmente a partir do locus Rosa26 9. Nós descrevemos um método para cultura da pele e capturar imagens embrionárias usando um microscópio confocal invertido. É adaptado forma do método original descrito em Mort et al. (2010) 6. O presentemétodo permite a formação de imagens de um formato de 6 poços e elimina a dependência de membranas Nuclepore e em matrigel para suportar a cultura. Em vez disso, utilizando um pequeno bloco de agarose a 1% para estabilizar a pele embrionário. Removendo a dependência de matrigel é importante, especialmente em situações em que a resposta da pele embrionário a factores de crescimento solúveis é o foco de estudo. Nosso método original já foi usado para ganhar novos insights sobre o desenvolvimento melanoblasto 10-13 e nós antecipamos que as melhorias que descrevemos aqui vai torná-lo mais poderoso como uma técnica experimental, especialmente onde várias culturas paralelas são uma exigência.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 são a crista neural precursores de melanócitos derivados; as células responsáveis ​​pela produção do pigmento na pele e no cabelo. Melanoblastos migra através da epiderme do embrião onde subsequentemente colonizar o cabelo desenvolver folículos 1,2. Migração das células da crista neural é amplamente estudada in vitro, mas em métodos in vivo ainda não estão bem desenvolvidos, especialmente em sistemas de mamíferos. Uma alternativa é a utilização ex vivo de cultura organotípica 3-6. Cultura de rato embrionário pele requer a manutenção de uma interface ar-líquido (ALI) em toda a superfície do tecido de 3,6. Alta resolução de imagem de live-rato pele embrionária tem sido dificultada pela falta de um bom método que não só mantém este ALI, mas também permite que a cultura seja invertida e, portanto, compatível com lentes objetivas de distância de trabalho curta e microscópios mais confocal. Este artigo descreve as recentes melhorias para a metanfetaminaod que utiliza uma membrana permeável a gás para superar estes problemas e permitir alta resolução de imagem confocal de pele embrionário em cultura ex vivo 6. Ao utilizar um melanoblasto específica Cre-recombinase expressa linha de rato combinado com a linha de repórter R26YFPR que são capazes de marcar por fluorescência da população melanoblasto dentro destas culturas de pele. A técnica permite ao vivo de imagem de melanoblastos e observação de seu comportamento e interações com o tecido em que se desenvolvem. Os resultados representativos são incluídos para demonstrar a capacidade de viver-imagem seis culturas em paralelo.

A Figura 2 mostra resultados representativos de um experimento de lapso de tempo usando pele embrionária de Tyr :: CREB x embriões R26YFPR em E14.5. 6 culturas de pele embrionários foram fotografadas por microscopia confocal a cada 2 min durante 18 horas. O pacote ImageJ software de análise de imagem gratuito foi usado para analisar o comportamento dos melanoblastos YFP-rotulados nos 6 filmes. Os melanoblastos foram automaticamente rastreados usando o plugin wrMTrck desenvolvido po...

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

Descrevemos a dissecção e ex vivo cultura de rato pele embrionária. O sistema mantém uma cultura de interface ar-líquido através da superfície do tecido e permite imagens em um microscópio invertido. Melanoblastos, um componente da pele em desenvolvimento, são marcadas por fluorescência, permitindo o seu comportamento a ser observada por meio de microscopia confocal.

Ex vivo Cultura do Rato Embryonic pele e Live-imaging de Migração melanoblasto

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

ex-vivo-culture-mouse-embryonic-skin-live-imaging-melanoblast

Research

JoVE Journal

Biology

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

12.5K visualizações.  University of Edinburgh.  Descrevemos a dissecção e ex vivo cultura de rato pele embrionária. O sistema mantém uma cultura de interface ar-líquido através da superfície do tecido e permite imagens em um microscópio invertido. Melanoblastos, um componente da pele em desenvolvimento, são marcadas por fluorescência, permitindo o seu comportamento a ser observada por meio de microscopia confocal. 

Vídeo: 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 a Cultura de Embriões início pintainho ex vivo (Nova 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 ...

Biologia

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.

Imagens ao vivo da migração das células da glia no Disco Drosophila Eye Imaginal

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.

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia 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.

Imagens ao vivo de Drosophila melanogaster Embrionárias Migrações hemócitos

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.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

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 de Cultura Primária células epiteliais trompa de Falópio

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.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

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 Cultura para o Rato coração embrionário

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.

Rato Fetal Whole Intestino Sistema de Cultura para<em&gt; Ex Vivo</em&gt; Manipulação de vias de sinalização e tridimensional Imagens ao vivo de Desenvolvimento Villus

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.

Medindo a rigidez<em&gt; Ex Vivo</em&gt; Mouse aortas Usando Microscopia de Força Atômica

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