Los autores desean dar las gracias al equipo de Servicios Creativos de la Universidad de Missouri Centro de Apoyo Académico quien produjo este video. Este trabajo fue apoyado por el Eunice Kennedy Shriver National Institute of Child Development Humanos y de Salud de los Institutos Nacionales de Salud, el número de concesión HD055231

1. La disección de la placenta del ratón <ol><li> Configure parejas de apareamiento de ratones. </li><li> En cada uno de los días siguientes, para comprobar la presencia de un tapón copulatorio. El día en el que se detecta un enchufe se considera 0,5 días post-coito (dpc) 7. </li><li> Placentas puede ser recogida en la etapa deseada del embarazo, con una disección limpia de la placenta principio posible con el desarrollo de la placenta madura en d 10,5. Aquí, demostramos 14,5 dpc. En los volúmenes dados, 40-60 placentas en 10,5 dpc, y 10-20 en placentas 14,5 dpc dará lugar a una capa de células del trofoblasto claramente visible. </li><li> Sacrificar al ratón embarazada, y extirpar el útero, el corte en el cuello uterino, ya lo largo de la mesometrium. </li><li> Con unas tijeras afiladas, corte entre cada embrión (Figura 1A). </li><li> Utilizando dos pares de pinzas, sujete la pared uterina en el borde cortado, y tire de ellos, la eliminación del miometrio (Figura 1B)<html> . Un par de sierra, fórceps moderadamente finas (curvo o recto), más un par de pinzas muy finas (# 5, por ejemplo) se recomienda. </li><li> Frente a la placenta, abra la decidua con pinzas o tijeras, exponiendo placenta. Agarrando el tejido uterino con un par pinzas, fórceps deslizar otra debajo de la placenta se separe (Figura 1 C). Si el feto es necesario, independiente y membranas fetales de la placenta. No es posible eliminar todo el tejido decidual por disección sin perder también tejido de la placenta, pero el tejido decidual adicional (pálido) se puede quitar de la superficie de la placenta suavemente peeling con fórceps. Vamos a confiar en el gradiente de Percoll para una purificación adicional. </li><li> Coloque cada placenta disecado en 25 ml de hielo frío tampón de disociación en un tubo cónico de 50 ml hasta que todas las disecciones se han completado. En etapas posteriores, toscamente picada tejido de la placenta con unas tijeras o una cuchilla de afeitar antes de la transferencia en tampón de disociación. </li></ol>tle &quot;2&gt;. Aislamiento de células trofoblásticas <ol><li> Sin apretar la tapa cónica del tubo y colocarlo en un baño a 37 ° C durante 45-60 minutos. Mezclar la solución de disociación vigorosamente pipeteando aproximadamente cada 10 min. El tiempo total de digestión deben ser monitoreados de cerca y es probable que requiera un poco de ensayo y error para lograr una recuperación óptima de la célula. Los tejidos deben ser capaces de pasar a través de una pipeta de 10 ml, pero las piezas visibles del tejido se mantendrá. Overdigestion resultará en la supervivencia celular y la pobre eficiencia de plaqueo. La supervivencia celular óptima y eficiencia de plaqueo se producirá cuando grupos de unas pocas células están presentes, en lugar de cuando se completa disociación se logra. Por el contrario, underdigestion conducirá a la pureza reducido después de la separación de Percoll. Cuando el punto de digestión se ha alcanzado, tubo lugar en hielo para detener la actividad de colagenasa. </li><li> Pasar la solución a través de un filtro de células para eliminar los materiales no digeridos, y se centrifuga a 500 xg durante 5 minutos. </li><li> Para lavar, quitarsobrenadante y resuspender las células en 10 ml de solución de lavado. Centrifugar a 500 xg durante 5 minutos. </li><li> Durante la centrifugación, preparar la solución de Percoll por mezcla de 9,6 ml de Percoll, 13,4 ml solución de lavado y 1,1 ml de medio de 10 veces en un tubo de 199 ml 50 apropiado para centrifugación de alta velocidad. </li><li> Eliminar celular sobrenadante y resuspender el pellet en 2 ml de solución de lavado. </li><li> Añadir células a Percoll solución y centrifugar @ 30.000 xg durante 30 minutos a 4 ° C. </li><li> Retire el tubo de centrífuga sin alterar el gradiente. Para visualizar el gradiente, tubo lugar delante de una fuente de luz brillante. Localizar la banda trofoblasto (Figura 2), y aspirar con una pipeta, teniendo cuidado de no alterar la banda de glóbulos rojos por debajo. Volumen de la banda trofoblasto será 5-8 ml. </li><li> Transferir las células trofoblásticas a un tubo cónico de 50 ml. Añadir la solución de lavado a un volumen total de 45 ml. </li><li> Centrifugar a 500 xg durante 5 minutos. Retirar el sobrenadante y resuspender en la cultura medium (NCTC-135). Las células pueden ser cultivadas sobre Matrigel de capa fina o de plástico de cultivo de tejidos a 37 º C y 5% CO 2. En el día 10.5, cada placenta produce unas 50.000 células vivas, de las cuales el 80-90% son citoqueratina positiva. </li></ol> 3. Trofoblasto invasión de ensayo <ol><li> Añadir 1-2 ml medio de cultivo (NCTC-135) a cada cámara de matrigel y se incuba a 37 °, 5% de CO 2 durante 30-90 min. </li><li> Retire medio y añadir 1,5 ml x 10 5 células recién aisladas en medio de 2 ml a la cámara superior. Añadir 1 ml de medio de la cámara baja para cubrir la superficie inferior de membrana. </li><li> Células Cultura inserta durante 18-24 horas. </li><li> Células raspar y matrigel de la inserción utilizando un hisopo de espuma. </li><li> Enjuague la parte superior e inferior de la cámara en 2 ml de PBS. </li><li> Arreglo para el 5 min en 1 ml de paraformaldehído al 4% en PBS. </li><li> Stain llenando la cámara inferior con 400 l de DAPI (300 nM) y se incuba durante 30 min. </li><li> Lavar tres veces brevemente en PBS. </li><li&gt; Quitar las membranas de insertos con una hoja de bisturí afilado. </li><li> Añadir una gota de 10 ul de medio de montaje (50% de glicerol en PBS) se deslice. Utilizando pinzas, coloque la membrana en la caída. Añadir otra caída de 10 l de medio, entonces el cubreobjetos con cuidado. </li><li> Fotografía y recuento de células. Asegúrese de orientar la membrana apropiadamente (celda hacia arriba o celda lateral abajo) para el microscopio que se utiliza. Image J software (Institutos Nacionales de Salud) incluye una característica de recuento manual que se puede utilizar para este propósito, al igual que Adobe Photoshop o un contador manual. </li></ol> 4. Los resultados representativos <img src="/files/ftp_upload/3202/3202fig1.jpg" alt="Figura 1" /> Figura 1. Disección de la placenta del ratón. (A) El útero se elimina por corte a lo largo de los oviductos (amarillo), a lo largo de la superficie mesometrial, y el cuello uterino (la parte inferior). Conceptuses individuales pueden ser separados cortando con tijeras, como se muestra. (B) La uterino<html> pared se elimina mediante la colocación de dos fórceps en la superficie uterina corte, y rasgando a lo largo del lado anti-mesometrial, cerca del feto y lejos de la placenta. (C) La placenta se aísla luego sujetando con una pinza deslizante y otro entre la placenta y el tejido uterino. El cordón umbilical y otras membranas extraembrionarias entonces se puede separar de la placenta. <img src="/files/ftp_upload/3202/3202fig2.jpg" alt="Figura 2" /> Figura 2. Percoll separación de las células trofoblásticas. Después de la centrifugación, tres bandas principales será visible como se muestra en la historieta de la izquierda, y una fotografía de la derecha. La banda superior contiene restos celulares y fibroblastos y será más evidente. Un apretado (rojo) banda cerca de la parte inferior que contiene las células rojas de la sangre será visible justo debajo de la banda de trofoblasto difusa. Agregados celulares es probable que se ve en la banda trofoblasto, como en los dos se muestra justo a la izquierda de la flecha en la fotografía. ove_content &quot;&gt; <img src="/files/ftp_upload/3202/3202fig3.jpg" alt="Figura 3" /> Figura 3. Células invasoras trofoblasto (flecha blanca) pasará a través de la capa de matrigel y los poros de la membrana, aparece en la superficie inferior de la membrana. Los poros son también visibles (negro punta de flecha). El número de células sobre la superficie de la membrana entera o un área de muestra representativa puede ser contada. Una imagen en escala de grises de fluorescencia DAPI nuclear se ha invertido para mejorar la visualización de las células. <img src="/files/ftp_upload/3202/3202fig4.jpg" alt="Figura 4" /> Células Figura 4. Trofoblasto puede ser identificada, y la pureza de la población aislada evaluados, por inmunotinción para citoqueratina 7 (centro, magenta). Las células se tiñen con DAPI (arriba, azul). Imagen fusionada inferior.

<ol>
	<li>Thordarson, G., Folger, P., Talamantes, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+placental+cell+culture+system+for+studying+the+control+of+mouse+placental+lactogen+II+secretion.">Development of a placental cell culture system for studying the control of mouse placental lactogen II secretion.</a> Placenta. 8, 573-585 (1987).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods. Mol. Med. 121, 203-217 (2006).</li><li>Kliman, H. J., Nestler, J. E., Sermasi, E., Sanger, J. M., Strauss, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+in+vitro+differentiation+of+cytotrophoblasts+from+human+term+placentae.">Purification, characterization, and in vitro differentiation of cytotrophoblasts from human term placentae.</a> Endocrinology. 118, 1567-1582 (1986).</li><li>Blaschitz, A., Weiss, U., Dohr, G., Desoye, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+reaction+patterns+in+first+trimester+placenta:+implications+for+trophoblast+isolation+and+purity+screening.">Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening.</a> Placenta. 21, 733-741 (2000).</li><li>Librach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=92-kD+type+IV+collagenase+mediates+invasion+of+human+cytotrophoblasts.">92-kD type IV collagenase mediates invasion of human cytotrophoblasts.</a> J. Cell. Biol. 113, 437-449 (1991).</li><li>Schulz, L. C., Widmaier, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+leptin+on+mouse+trophoblast+cell+invasion.">The effect of leptin on mouse trophoblast cell invasion.</a> Biol. Reprod. 71, 1963-1967 (2004).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

No tenemos nada que revelar.

En este video se demuestra el aislamiento de las células trofoblásticas de la placenta del ratón. Luego mostramos un ensayo in vitro para la invasión del trofoblasto. Usando este procedimiento, una gran parte, pero no totalmente, la población pura de células trofoblásticas se puede conseguir. Si se requiere mayor pureza, separación magnética perla o citometría de flujo se pudo realizar, como se ha descrito para las células trofoblásticas humanas primarias. La longitud adecuada de la etapa de colagenasa disoc...

Specific reagents and equipment:
Wash Solution (filter sterilize after mixing)
<table border='1'>
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>50ml</td>
 <td>1X</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>2.383 g</td>
 <td>0.02M</td>
 </tr>
 <tr>
 <td>sodium bicarbonate</td>
 <td>0.42 g</td>
 <td>0.01M</td>
 </tr>
 <tr>
 <td>Penicllin-Streptomycin</td>
 <td>5 mL</td>
 <td>100 U- 100 &mu;g /ml</td>
 </tr>
 <tr>
 <td>Sterile dH20</td>
 <td>to 500 mL</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

Dissociation solution (make fresh and filter sterilize)
<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Amount</td>
 <td>Final concentration</td>
 </tr>
 <tr>
 <td>Wash Solution</td>
 <td>100 mL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>1 vial (2000 U)</td>
 <td>20 U/mL</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>100 mg</td>
 <td>125 digestion units/mL</td>
 </tr>
 </tbody>
</table>


<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Collagenase</td>
 <td>Sigma</td>
 <td>C9891</td>
 <td>This is Clostridium collagenase.
Bovine pancreatic collagenase would likely work as well</td>
 </tr>
 <tr>
 <td>NCTC-135</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>Add FBS to 10%</td>
 </tr>
 <tr>
 <td>Matrigel invasion chambers</td>
 <td>BD Biosciences</td>
 <td>354481</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10x Medium 199</td>
 <td>Sigma</td>
 <td>M9163</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>Sigma</td>
 <td>D4263</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>100 &mu;m Cell strainer</td>
 <td>Fisher</td>
 <td>22363549</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Antibody to cytokeratin 7</td>
 <td>DAKO</td>
 <td>M7018</td>
 <td>Used at 1:100 dilution</td>
 </tr>
 </tbody>
</table>

Equipment Standard cell culture equipment including a CO2 incubator is required. In addition, a centrifuge rotor capable of spinning 50 ml tubes at 30,000 x g is needed. Note that this exceeds the maximum speed of most conical centrifuge tubes and will likely require an Oak Ridge style tube.

isolation of primary mouse trophoblast cells and trophoblast invasion assay

La placenta es responsable del transporte de nutrientes, gases y factores de crecimiento para el feto, así como la eliminación de los desechos. Por lo tanto, los defectos en el desarrollo placentario tienen importantes consecuencias para el feto y la madre, y son una causa principal de la letalidad embrionaria. El principal tipo de células de la parte fetal de la placenta es el trofoblasto. Las células primarias de ratón trofoblasto placentario son una herramienta útil para el estudio de desarrollo de la placenta normal y anormal, y a diferencia de las líneas celulares, se pueden aislar y utilizar para estudiar trofoblasto en etapas específicas del embarazo. Además, los cultivos primarios de trofoblasto de ratones transgénicos pueden ser utilizados para estudiar la función de genes particulares en células de la placenta. El protocolo presentado aquí se basa en la descripción por Thordarson et al. 1, en ​​el que se utiliza un gradiente de Percoll para obtener una población de células relativamente puro trofoblasto de la placenta aislados de ratón. Es similar a utilizar el más ampliamentemétodos para 2-3 d aislamiento humano trofoblasto celular. Pureza puede ser evaluada por tinción inmunocitoquímica de las células aisladas para la citoqueratina 7 4. Aquí, las células aisladas se analizan entonces usando un ensayo de invasión de Matrigel para evaluar la invasividad del trofoblasto in vitro 5-6. Las células invadidas se analizaron mediante inmunocitoquímica y se tiñeron para el recuento.

Watch this Scientific Journal Video about Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay at JoVE.com

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

En este protocolo, se describe la disección de placenta de ratón en el embarazo D10.5, seguido por el aislamiento de las células trofoblásticas utilizando un gradiente de Percoll. A continuación, demostrar el uso de las células aisladas en un ensayo de invasión de Matrigel.

El aislamiento de células primarias de ratón trofoblasto y ensayo de invasión trofoblasto

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

isolation-primary-mouse-trophoblast-cells-trophoblast-invasion

Research

JoVE Journal

Biology

23.7K vistas.  University of Missouri. En este protocolo, se describe la disección de placenta de ratón en el embarazo D10.5, seguido por el aislamiento de las células trofoblásticas utilizando un gradiente de Percoll. A continuación, demostrar el uso de las células aisladas en un ensayo de invasión de Matrigel.

Video: Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivación de células madre de ratón a partir de blastocistos Trofoblasto

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Investigación

Biología

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Aislamiento y cultivo primario de células hepáticas de ratas

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

El aislamiento de las células inmunes de tumores primarios

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Aislamiento y cultivo de ratón primarias células acinares pancreáticas

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robusto PESCADO ADN 3D utilizando sondas marcadas directamente

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

Aislamiento de Miofibroblastos primarias de ratón y el tejido del colon humano

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for&#160;tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four&#160;passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Aislamiento, caracterización y alto rendimiento análisis de flujo extracelular de ratón células epiteliales tubulares renales primarias

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of&#160;chronic kidney disease (CKD). ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Colección de células únicas de células trofoblastas en embriones humanos en fase de implantación

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...