We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

NOTA: Los experimentos con animales se realizaron de acuerdo con los Institutos Nacionales de Salud de Guía para el Cuidado y Uso de Animales de Laboratorio. El Comité de Experimentación Animal de la Facultad de Medicina de la Universidad de Tohoku aprobó los procedimientos experimentales descritos en el presente documento. 1. Anestesia y laparotomía <ol style="margin-left: 40px;"><li> Para la recolección de la sangre, use ratas libres de patógenos específicos Sprague-Dawley. Fast las ratas durante al menos 18 horas, mientras que el suministro de agua. Utilice jubilados ratas reproductoras masculinas a los 6-8 meses de edad (550-650 g) para la recogida de sangre. </li><li> Flujo 2,5-4,0% de isoflurano, que es un anestésico de inhalación, en una caja conectada a un aparato de anestesia para introducir la anestesia en 2,0-3,5 l / min durante 10 min. Traslado ratas en el cuadro para introducir la anestesia. <ol><li> Confirmar anestesia mediante la comprobación de la pérdida de la respuesta a la estimulación en las patas con fórceps. Cúbrase la nariz de la wi ratasth una máscara conectada a un aparato de anestesia para anestesiar profundamente las ratas durante la extracción de sangre. La concentración y el flujo se mantienen a 2,5-4,0% y 2,0-3,5 l / min, respectivamente. NOTA: halotano no se debe utilizar para la anestesia de inhalación porque los embriones cultivados en el suero que se recoge de ratas anestesiadas con este reactivo exhiben un retraso en el desarrollo embrionario en comparación con los embriones cultivados en el suero obtenido de ratas anestesiadas con isoflurano 22. </li></ol></li><li> Para evitar la contaminación de la piel, la desinfección de la piel mediante el vertido de etanol al 70% en el abdomen de la rata anestesiada. Recoger la piel desinfectada y la pared abdominal en la región inferior con un par de pinzas y cortar estas capas simultáneamente hacia la región del tórax con un par de tijeras grandes para exponer los órganos internos. Reflejar el intestino fuera de la cavidad abdominal con un par de pinzas. </li><li> Cortar la piel y la pared abdominal hacia las extremidades traseras con unpar de tijeras grandes para exponer aún más la región abdominal posterior. Reflejar la grasa extra fuera de la cavidad abdominal con un par de pinzas. </li><li> Recoger la grasa visceral en la línea media usando dos pares de pinzas y dividir la grasa cuidadosamente en una dirección paralela para exponer la aorta abdominal. Repita este procedimiento para dividir la grasa visceral en una dirección longitudinal para identificar fácilmente la bifurcación de la aorta abdominal. Además, la aorta es pulsátil y se puede distinguir de la vena cava adyacente utilizando esta característica. </li><li> Recoge la grasa alrededor de la aorta abdominal y la bifurcación con cuidado con dos pares de pinzas y se separa la grasa en la aorta abdominal. </li></ol> 2. Extracción de sangre <ol style="margin-left: 40px;"><li> Con cuidado, inserte la punta de una aguja 21 conectada a una jeringa de 20 ml inmediatamente craneal a la bifurcación de la aorta, con el bisel en una dirección hacia abajo. Sostenga la jeringa con unamano, y tire la jeringa lentamente para recoger 15 ml de sangre de una rata macho. </li><li> Retire la aguja de la jeringa, y verter la sangre en dos tubos helados de 10 ml estériles de prueba (Figura 1A). Mantener los tubos en hielo hasta la centrifugación para retrasar la formación del coágulo de sangre. Reducir el tiempo de centrifugación por centrifugación varios tubos al mismo tiempo. </li><li> La eutanasia a la rata anestesiada por toracotomía y cortar el corazón o la decapitación. </li></ol> 3. Rata Preparación de suero <ol style="margin-left: 40px;"><li> Centrifugar la sangre recogida durante 5 min a 1200 x g y temperatura ambiente (TA) para separar la sangre en las capas superior e inferior (Figura 1C). </li><li> Mantener los tubos a 4 º C durante 1,5 a 2 horas para arreglar el coágulo de fibrina. </li><li> Levante el coágulo de fibrina en la capa superior con un par de pinzas curvas, y exprimir el coágulo de fibrina para separar el suero. A continuación, pulse el coágulo de fibrina con las pinzas curvas enfrentan downwards cerca de la interfaz entre las dos capas (Figura 1D). </li><li> Repita el paso 3.3 para separar aún más el coágulo de fibrina. </li><li> Centrifugar los tubos de ensayo durante 5 min a 1200 xg y RT (Figura 1E). </li><li> Transferir cuidadosamente el suero en un tubo estéril de 15 ml utilizando una pipeta estéril en un gabinete de flujo laminar de aire. </li><li> Centrifugar el suero de rata recogido durante 5 min a 1200 xg y RT para eliminar cualquier resto de células rojas. </li><li> Reúnase el suero con cuidado en un nuevo tubo de 15 ml esterilizados utilizando una pipeta estéril para evitar la contaminación por los glóbulos rojos residuales en la parte inferior del tubo (Figura 1F). </li><li> Incubar el suero en un baño de agua a 56 ° C durante 30 minutos para inactivar el sistema de complemento. </li><li> Enfriar los tubos de suero de rata a RT en el gabinete de flujo de aire laminar. El suero debe ser dividió en alícuotas a 10 ml en tubos estériles individuales. Guarde los tubos a -20 ° C hasta su uso (Figura 1H). </li></ol>

<ol>
	<li>New, D. A. T., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction.">Introduction.</a> Mammalian Postimplantation Embryos. A Practical Approach. , 1-14 (1990).</li><li>Eto, K., Takakubo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+development+of+rat+embryos+in+culture+during+the+period+of+craniofacial+morphogenesis.">Improved development of rat embryos in culture during the period of craniofacial morphogenesis.</a> J Craniofac Genet Dev Biol. 5, 351-355 (1985).</li><li>Takahashi, M., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+method+of+rodent+whole+embryo+culture+using+the+rotator-type+bottle+culture+system.">The method of rodent whole embryo culture using the rotator-type bottle culture system.</a> J Vis Exp. (42), e2170 (2010).</li><li>New, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+embryo+culture,+teratogenesis,+and+the+estimation+of+teratologic+risk.">Whole embryo culture, teratogenesis, and the estimation of teratologic risk.</a> Teratology. 42, 635-642 (1990).</li><li>Matsuo, 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+mutation+in+the+Pax-6+gene+in+rat+small+eye+is+associated+with+impaired+migration+of+midbrain+crest+cells.">A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells.</a> Nat Genet. 3, 299-304 (1993).</li><li>Nomura, T., Holmberg, J., Frisen, J., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6-dependent+boundary+defines+alignment+of+migrating+olfactory+cortex+neurons+via+the+repulsive+activity+of+ephrin+A5.">Pax6-dependent boundary defines alignment of migrating olfactory cortex neurons via the repulsive activity of ephrin A5.</a> Development. 133, 1335-1345 (2006).</li><li>Yamamoto, M., 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=Nodal+antagonists+regulate+formation+of+the+anteroposterior+axis+of+the+mouse+embryo.">Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo.</a> Nature. 428, 387-392 (2004).</li><li>Inoue, 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=Role+of+cadherins+in+maintaining+the+compartment+boundary+between+the+cortex+and+striatum+during+development.">Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development.</a> Development. 128, 561-569 (2001).</li><li>Takahashi, M., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6+regulates+specification+of+ventral+neurone+subtypes+in+the+hindbrain+by+establishing+progenitor+domains.">Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains.</a> Development. 129, 1327-1338 (2002).</li><li>Calegari, F., Haubensak, W., Yang, D., Huttner, W. B., Buchholz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+RNA+interference+in+postimplantation+mouse+embryos+with+endoribonuclease-prepared+short+interfering+RNA.">Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA.</a> Proc Natl Acad Sci U S A. 99, 14236-14240 (2002).</li><li>Takahashi, M., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferring+genes+into+cultured+mammalian+embryos+by+electroporation.">Transferring genes into cultured mammalian embryos by electroporation.</a> Dev Growth Differ. 50, 485-497 (2008).</li><li>Pryor, S. E., Massa, V., Savery, D., Greene, N. D., Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension+analysis+in+mouse+whole+embryo+culture.">Convergent extension analysis in mouse whole embryo culture.</a> Methods Mol Biol. 839, 133-146 (2012).</li><li>Kikkawa, 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=Dmrta1+regulates+proneural+gene+expression+downstream+of+Pax6+in+the+mammalian+telencephalon.">Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon.</a> Genes Cells. 18, 636-649 (2013).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240, 237-246 (2001).</li><li>Turnbull, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+backscatter+microscopy+of+mouse+embryos.">Ultrasound backscatter microscopy of mouse embryos.</a> Methods Mol Biol. 135, 235-243 (2000).</li><li>Pierfelice, T. J., Gaiano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided+microinjection+into+the+mouse+forebrain+in+utero+at+E9.5.">Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.</a> J Vis Exp. (45), e2047 (2010).</li><li>Quinlan, G. A., Khoo, P. L., Wong, N., Trainor, P. A., Tam, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+grafting+and+labeling+in+postimplantation+mouse+embryos.">Cell grafting and labeling in postimplantation mouse embryos.</a> Methods Mol Biol. 461, 47-70 (2008).</li><li>Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R., Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roller+culture+of+postimplantation+embryos.">Roller culture of postimplantation embryos.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , 237-241 (2003).</li><li>Garcia, M. D., Udan, R. S., Hadjantonakis, A. K., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+for+culturing+mouse+embryos.">Preparation of rat serum for culturing mouse embryos.</a> Cold Spring Harb Protoc. 2011, 391-393 (2011).</li><li>Brown-Woodman, P. D., Ritchie, H. E., Korabelnikoff, A., Emmanuel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+ether+with+alternate+volatile+anesthetics+for+collection+of+rat+serum+used+in+embryo+culture.">Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture.</a> Toxicol In Vitro. 18, 719-724 (2004).</li><li>Gray, J., Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tube+closure+in+mouse+whole+embryo+culture.">Neural tube closure in mouse whole embryo culture.</a> J Vis Exp. (56), e3132 (2011).</li><li>Machado, C. B., 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=Reconstruction+of+phrenic+neuron+identity+in+embryonic+stem+cell-derived+motor+neurons.">Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.</a> Development. 141, 784-794 (2014).</li><li>Glanville-Jones, H. C., Woo, N., Arkell, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+whole+embryo+culture+with+commercially+available+reagents.">Successful whole embryo culture with commercially available reagents.</a> Int J Dev Biol. 57, 61-67 (2013).</li><li>Ornoy, A., Yacobi, S., Yaffee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+culture+of+11.5-day-old+rat+embryos+in+DMEM/F12+and+20%+fetal+bovine+serum.">A simple method of culture of 11.5-day-old rat embryos in DMEM/F12 and 20% fetal bovine serum.</a> J Anat. 203, 419-423 (2003).</li><li>Moore-Scott, B. A., Gordon, J., Blackburn, C. C., Condie, B. G., Manley, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+serum-free+in+vitro+culture+technique+for+midgestation+mouse+embryos.">New serum-free in vitro culture technique for midgestation mouse embryos.</a> Genesis. 35, 164-168 (2003).</li><li>Kalaskar, V. K., Lauderdale, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Embryonic+Development+in+a+Serum-free+Whole+Embryo+Culture+System.">Mouse Embryonic Development in a Serum-free Whole Embryo Culture System.</a> J Vis Exp. (85), e50308 (2014).</li></ol>

No conflicts of interest are declared.

Resultados reproducibles en experimentos WEC dependen de la utilización de alta calidad IC suero de rata y técnica de disección precisa embrión 3. No acortar el período de ayuno antes de la recogida de sangre, porque el ayuno es necesario normalizar las concentraciones de glucosa en la sangre. Las ratas hembras no deben ser utilizados para la preparación de suero debido a los niveles hormonales cambian en animales hembras de acuerdo con el ciclo estral, y tales cambios en las hormonas estradiol no son a...

Una variedad de modelos animales se utilizan en la biología del desarrollo para investigar los mecanismos de desarrollo a nivel molecular y celular. Por ejemplo, las especies de anfibios y aves han sido ampliamente utilizados como animales modelo clásico que son adecuados para la manipulación directa de embriones debido a que estos embriones se desarrollan fuera de la madre. En contraste con estos animales, los embriones de mamíferos crecen en el útero de la madre, y el crecimiento en etapas posteriores es crucialmente dependiente de la función del útero. Por lo tanto, normalmente es difícil de manipular directamente los embriones de mamíferos tales como los del ratón y la rata en las primeras etapas. En la década de 1960, Denis Nueva estableció un cultivo de embriones de mamíferos toda técnica (WEC), utilizando aparatos de WEC con un suministro de oxígeno continuo y control de calor 1. En WEC, ratón y rata embriones pueden crecer ex vivo, (es decir, fuera del útero). Aunque la técnica de WEC fue utilizado a menudo en teratología, mediante la adición de varios chemicotros compuestos en el medio de cultivo, esta técnica también se ha utilizado en varios estudios de biología del desarrollo para examinar los mecanismos de desarrollo únicos en mamíferos 2-4. Por ejemplo, el CME se combina con otras técnicas, como el etiquetado de las células, en los de tipo salvaje y mutante embriones mediante el uso de tinte fluorescente 5, el trasplante de células 6, y la introducción de genes a través de lipofección y electroporación 7 8-13. Recientemente, la manipulación en el útero se ha utilizado para analizar los procesos de desarrollo en embriones de roedores en etapas posteriores y se ha combinado con técnicas de electroporación 14-16. Sin embargo, estas técnicas no son adecuadas para la manipulación de embriones postimplante y mitad de la gestación debido a dificultades para alcanzar la inyección local exacta de la solución de ADN en embriones en las primeras etapas. Aunque el trasplante de células guiada por ultrasonido y la inyección de vectores virales en embriones tempranos (es decir,, Se ha informado de E8.5-E-9.5 en el ratón) en el útero previamente 17,18, se requieren excelentes habilidades para llevar a cabo estos experimentos con un alto índice de éxito. Por lo tanto, el CME con alta accesibilidad ex vivo tiene ventajas con respecto a la manipulación de embriones de ratón y rata. Inmediatamente se centrifugó (IC) de suero de rata preparado a partir de ratas macho se utiliza a menudo para el medio WEC. Cuando los embriones se cultivan en la fase después de la implantación (es decir, antes de E8.0 en el ratón o E10.0 en la rata), una mezcla de medio sintético y suero de rata IC se utiliza a menudo como un medio para la WEC 19. Sin embargo, a los embriones de cultivo a la mitad de la gestación (es decir., E8.0-12.5 en embriones de ratón E10.0-E14.5 o en embriones de rata), 100% de suero debe ser usado como un medio porque no hay medios de comunicación alternativa disponible actualmente permite embriones para crecer normalmente in vitro durante más de 2 días. La preparación de suero de rata de alta calidad esun paso crítico para lograr la reproducibilidad en los experimentos de WEC. En comparación con retraso centrifugación, IC tiene el beneficio de reducir la hemólisis cuando el suero se recogió por apretar el coágulo de fibrina, porque la mayoría de las células rojas de la sangre ya se han separado del coágulo de fibrina. Como suero de rata hemolítica no apoya el crecimiento normal de los embriones de rata y ratón, la preparación de suero utilizando centrifugación inmediata es preferible a la preparación usando retraso centrifugación. Nuestro protocolo contiene dos pasos adicionales en comparación con otros protocolos 18-20 (es decir., El almacenamiento de la sangre recogida en hielo antes de la primera centrifugación y el mantenimiento de las muestras de sangre recogidas durante 2 horas a 4 ° C después de la primera centrifugación). La etapa anterior puede retrasar la formación del coágulo de sangre, y el segundo paso promueve la solidificación del coágulo de fibrina para una fácil compresión. Por lo tanto, nuestro protocolo se puede utilizar por los principiantes. Sin embargo, la reproducción de la recogida de sangre y sueroextracción con precisión por una simple referencia a los libros de protocolo es extremadamente difícil 19-21. En este artículo de vídeo, demostramos nuestro protocolo estándar para la preparación de suero de rata óptima IC, que incluye la recogida de sangre de la aorta abdominal de las ratas macho y la extracción del suero por centrifugación.

<table border="0" cellpadding="0" cellspacing="0" width="892">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:401px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>For anesthesia of rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Large scissors</td>
			<td>Napox</td>
			<td>B-7H</td>
			<td>Stelized scissors for cutting the skin and muscle of rats.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Curved forceps</td>
			<td>Napox</td>
			<td>A-3-2</td>
			<td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sprague-Dawley (SD) rat</td>
			<td>Charles Rivers Laboratories</td>
			<td></td>
			<td>Retired male rats from colony in the lab or purchased retired male rats.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe (20 ml)</td>
			<td>TERUMO</td>
			<td>SS-29ESZ</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Needle (21G x 5/8&#34;)</td>
			<td>TERUMO</td>
			<td>NN-2116R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile test (spitz) tube (10 ml)</td>
			<td>ASIAKIZAI</td>
			<td style="width:119px;">1101C000B-10</td>
			<td>For collection of boold</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable pipette</td>
			<td>Eiken Chemical</td>
			<td>CD2000 No.4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterilie disposable tube (15 ml)&#160;</td>
			<td>Falcon</td>
			<td>2196</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

La Figura 1 muestra los resultados representativos de los procedimientos descritos para la separación de células de la sangre y suero. Por lo general obtenemos 15 ml de sangre de una rata macho retirado (Figura 1A). Por centrifugación, la sangre recogida se puede separar en una capa superior que contiene el suero y el coágulo de fibrina, que se indica con una flecha, y una capa inferior que contiene las células de la sangre (Figura 1C). Es importante destacar que l...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparación de suero de rata Adecuado para mamíferos Whole cultivo de embriones

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

13.7K vistas.  Jichi Medical University. Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Video: Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Cultura Cortar organotípicos de los cerebros de ratas E18

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Investigación

Biología

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultivo primario de miocitos adultos de corazón de rata

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

Preparación y Cultura de la lente explantes Rata epiteliales para el estudio de la diferenciación terminal

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Un Ex-ovo</emPollo> Embrión de cultivo apropiado para aplicaciones de imagen y sistema de Microcirugía

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

El método de cultivo de embriones de roedores enteros utilizando el sistema de tipo de botella rotador Cultura

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Preparación de los tendones Cola de Rata de Estudios biomecánicos y Mechanobiological

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

La ampliación de los cultivos celulares de mamíferos con un frasco de varias capas de Nueva

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Preparación de las células embrionarias de fibroblastos de ratón adecuados para el cultivo de células estaminales embrionarias humanas pluripotentes inducidas, y

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embriones de ratón con el desarrollo en todo un sistema de cultivo de embriones sin suero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Preparativos Piso en observación y análisis de embrión de pez cebra muestras teñidas por todo el montaje<em&gt; In situ</em&gt; La hibridación

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...