Os autores gostariam de agradecer à equipa Serviços Criativos da Universidade de Missouri Centro de Apoio Acadêmico, que produziu este vídeo. Este trabalho foi financiado pelo Eunice Kennedy Shriver National Institute of Child Development Saúde e Humanos do Instituto Nacional de Saúde, subvenção número HD055231

1. Dissecção da placenta do mouse <ol><li> Configure pares de acasalamento dos ratos. </li><li> Em cada um dos dias seguintes, para verificar a presença de um tampão copulatório. O dia em que é detectada uma ficha é considerada 0,5 dias pós-coito (dpc) 7. </li><li> Placentas podem ser obtidas na fase desejada da gestação, com uma dissecção limpa do início placenta possível com o desenvolvimento da placenta madura em d 10,5. Aqui, nós demonstramos 14,5 dpc. Nos volumes indicados, 40-60 placentas de 10,5 dpc, e 10-20 placentas de 14,5 dpc produzirá uma camada de células de trofoblasto claramente visível. </li><li> Eutanásia do rato prenhe, e remover o útero, o colo do útero em corte, e ao longo da mesométrio. </li><li> Com uma tesoura afiada, corte entre cada concepto (Figura 1A). </li><li> Utilizando dois pares de pinças, segure a parede uterina no bordo de corte, e separar, a remoção do miométrio (Figura 1B)<html> . Um par serrilhada, fórceps moderadamente finas (curvo ou reto), além de um par de fórceps muito finas (# 5, por exemplo) é recomendado. </li><li> Em frente à placenta, abra a decídua com pinças ou tesouras, expondo placenta. Agarrando o tecido uterino com um par fórceps, deslize por baixo da outra pinça para separar a placenta (Figura 1C). Se o feto, necessário separar e da placenta. Não é possível remover todo o tecido decidual por dissecção sem também perder tecido placentário, mas o tecido decidual adicional (pálido) pode ser removida da superfície da placenta suavemente por descamação com um fórceps. Contamos com o gradiente de Percoll para posterior purificação. </li><li> Coloque cada placenta dissecada em 25 ml de tampão gelada dissociação em um tubo de 50 ml cônico até que todas as dissecações estão completos. Em fases posteriores, grosseiramente picada tecido placentário com tesoura ou lâmina de barbear antes de se transferir para o buffer dissociação. </li></ol>tle &quot;&gt; 2 Isolamento. das células trofoblásticas <ol><li> Vagamente tubo tampa cônica e coloque em banho a 37 ° C por 45-60 minutos. Misturar a solução de dissociação por pipetagem vigorosa aproximadamente a cada 10 min. O tempo de digestão total deve ser acompanhada de perto e provavelmente vai exigir alguma tentativa e erro para conseguir a recuperação celular ideal. Os tecidos devem ser capazes de ser passado através de uma pipeta de 10 ml, mas pedaços visíveis de tecido permanecerá. Overdigestion irá resultar em sobrevivência celular pobre e a eficiência de plaqueamento. A sobrevivência das células e eficiência de plaqueamento óptima ocorrerão quando aglomerados de algumas células estão presentes, e não quando a dissociação completa seja alcançada. Por outro lado, underdigestion levará a pureza reduzida após separação percoll. Quando o ponto de digestão tiver sido atingido, o tubo de colocar em gelo para parar a actividade da colagenase. </li><li> Filtrar a solução através de um filtro celular para remover os materiais não digeridos, e centrifugar @ 500 xg durante 5 minutos. </li><li> Para lavar, removasobrenadante e ressuspender as células em 10 ml de solução de lavagem. Centrifugar @ 500 xg durante 5 minutos. </li><li> Durante a centrifugação, preparar uma solução de Percoll, misturando 9,6 ml de Percoll, 13,4 ml de solução de lavagem e de 1,1 ml de meio de 10x 199 em um tubo de 50 ml, apropriado para centrifugação de alta velocidade. </li><li> Remover o sobrenadante e ressuspender o sedimento de células em 2 ml de solução de lavagem. </li><li> Adicionar células a solução de Percoll e centrifuga @ 30000 xg durante 30 minutos a 4 ° C. </li><li> Retire o tubo de centrífuga, sem perturbar o gradiente. Para visualizar o gradiente tubo lugar, em frente de uma fonte de luz brilhante. Localizar a banda trofoblasto (Figura 2) e, com uma pipeta de aspiração, tendo o cuidado de não perturbar a banda de células vermelhas do sangue a seguir. Volume da banda trofoblasto será 5-8 ml. </li><li> Transferir células trofoblásticas para um tubo cónico de 50 ml. Adicionar solução de lavagem para um volume total de 45 ml. </li><li> Centrifugar @ 500 xg durante 5 minutos. Remover o sobrenadante e ressuspender em cultura medihm (NCTC-135). As células podem ser cultivadas em camada fina matrigel ou de plástico de cultura de tecidos a 37 ° C e 5% CO 2. No dia 10,5, cada placenta produz cerca de 50.000 células vivas, dos quais 80-90% são citoqueratina positivas. </li></ol> 3. Ensaio de invasão do trofoblasto <ol><li> Adicionar 1-2 ml meio de cultura (NCTC-135) para cada uma das câmaras de matrigel e incubar a 37 ° C, 5% CO 2 por 30-90 min. </li><li> Remover meio e adicionar 1,5 ml x 10 5 células isoladas de fresco em meio de 2 ml de câmara superior. Adicionar 1 ml de meio de câmara inferior para cobrir a superfície inferior da membrana. </li><li> As células de cultura em inserções durante 18-24 horas. </li><li> Células raspar e Matrigel da inserção utilizando um cotonete de espuma. </li><li> Enxaguar o topo e fundo da câmara, em 2 ml de PBS. </li><li> Fix, durante 5 min em 1 ml de paraformaldeído 4% em PBS. </li><li> Stain de enchimento da câmara inferior com 400 ul de DAPI (300 nM), e incubar durante 30 min. </li><li> Lavar três vezes brevemente em PBS. </li><li&gt; Remover as membranas a partir de pastilhas com uma lâmina afiada bisturi. </li><li> Adicionar uma gota de 10 ul de meio de montagem (glicerol a 50% em PBS) a deslizar. Utilizando uma pinça, colocar membrana em queda. Adicionar outra gota de 10 ul de meio, em seguida, cuidadosamente lamela. </li><li> Células fotografia e contagem. Certifique-se de orientar adequadamente a membrana (célula voltada para cima ou célula-side down) para o microscópio que está sendo usado. Imagem de J software (National Institutes of Health) inclui uma característica de contagem manual que pode ser utilizado para este fim, como pode o Adobe Photoshop ou um contador manual. </li></ol> 4. Resultados representativos <img src="/files/ftp_upload/3202/3202fig1.jpg" alt="Figura 1" /> Figura 1. Dissecção da placenta de rato. (A) O útero é removido através de corte ao longo dos ovidutos (amarelo), ao longo da superfície mesometrial, e no colo do útero (parte inferior). Conceptos individuais podem então ser separadas por corte com uma tesoura imediata. (B) O uterino<html> parede é removido colocando duas pinças na superfície de corte do útero, e ao rasgamento ao longo do lado anti-mesometrial, perto do feto e para longe da placenta. (C) A placenta é então isolado por meio de agarramento com uma pinça deslizante e uma outra entre a placenta e tecido uterino. Cordão umbilical e outras membranas extra-embrionário pode então ser separado da placenta. <img src="/files/ftp_upload/3202/3202fig2.jpg" alt="Figura 2" /> Figura 2. Percoll separação das células trofoblásticas. Após a centrifugação, três bandas principais será visível, como mostrado em desenhos animados à esquerda, e uma fotografia à direita. A banda superior contém detritos celulares e os fibroblastos e será mais aparente. Uma banda (vermelho) apertado perto da parte inferior que contém as células vermelhas do sangue será visível logo abaixo da banda difusa trofoblasto. Agregados celulares provavelmente visto na banda de trofoblasto, como nos dois mostrada à esquerda da seta na fotografia. ove_content &quot;&gt; <img src="/files/ftp_upload/3202/3202fig3.jpg" alt="Figura 3" /> Figura 3. Células trofoblásticas invasoras (seta branca) vai passar através da camada de matrigel e os poros da membrana, que aparece sobre a superfície inferior da membrana. Os poros também são visíveis (seta preta). O número de células na superfície da membrana inteira ou uma área de amostra representativa pode ser contado. Uma imagem em tons de cinza de coloração fluorescente DAPI nuclear foi invertido para melhorar a visualização das células. <img src="/files/ftp_upload/3202/3202fig4.jpg" alt="Figura 4" /> As células trofoblásticas Figura 4. Podem ser identificados, e a pureza da população isolada avaliada, por imunocoloração para citoqueratina 7 (centro, magenta). As células são contrastadas com DAPI (topo, azul). Incorporada imagem de baixo,.

<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>

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Neste vídeo, demonstramos o isolamento de células trofoblásticas da placenta de rato. Depois, mostramos um ensaio in vitro para a invasão do trofoblasto. Utilizando este procedimento, uma grande parte, mas não totalmente, população pura de células trofoblásticas pode ser obtida. Se a pureza é necessária mais, separação magnética ou grânulo de citometria de fluxo pode ser realizada, como já foi descrito para as células trofoblásticas humanas primárias. O comprimento adequado do passo de dissociação c...

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

A placenta é responsável para o transporte de nutrientes, gases e factores de crescimento para o feto, bem como a eliminação de desperdícios. Assim, defeitos de desenvolvimento da placenta ter importantes conseqüências para o feto ea mãe, e são uma das principais causas de letalidade embrionária. O principal tipo de célula da porção fetal da placenta é o trofoblasto. Primários de rato trofoblasto da placenta é uma ferramenta útil para o estudo do desenvolvimento normal e anormal da placenta, e ao contrário de linhas de células, pode ser isolado e utilizado para estudar trofoblasto em fases específicas da gravidez. Além disso, a culturas primárias de trofoblasto de ratinhos transgénicos podem ser utilizados para estudar o papel de genes específicos em células da placenta. O protocolo aqui apresentado é baseado na descrição por Thordarson et al. 1, no qual um gradiente de Percoll é usado para obter uma população de células relativamente puro trofoblasto da placenta de rato isolados. Ele é similar ao utilizar mais amplamented humano métodos para isolamento 2-3 trofoblasto célula. A pureza pode ser avaliada por coloração imunocitoquímica das células isoladas para citoqueratina 7 4. Aqui, as células isoladas são então analisadas utilizando um ensaio de invasão para avaliar a capacidade de invasão de matrigel trofoblasto in vitro 5-6. As células invadidas são analisados ​​por imunocitoquímica e coradas para contagem.

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

Neste protocolo, descreve-se a dissecção de placenta de rato sobre a gravidez d10.5, seguido de isolamento das células trofoblásticas, utilizando um gradiente de Percoll. Em seguida, demonstrar o uso de células isoladas, num ensaio de invasão de Matrigel.

Isolamento de células primário do mouse trofoblasto e Ensaio invasão do 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

23.4K visualizações.  University of Missouri. Neste protocolo, descreve-se a dissecção de placenta de rato sobre a gravidez d10.5, seguido de isolamento das células trofoblásticas, utilizando um gradiente de Percoll. Em seguida, demonstrar o uso de células isoladas, num ensaio de invasão de Matrigel.

Vídeo: 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.

Derivação de células-tronco do rato trofoblasto de blastocistos

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

Biologia

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%.

Isolamento e cultura primária de células hepáticas de ratos

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.

Isolamento de células imunes de tumores primários

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.

Isolamento e Cultura do mouse primárias células pancreáticas acinares

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.

Robust FISH DNA 3D utilizando sondas Diretamente rotuladas

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.

Isolamento de primárias Miofibroblastos de Mouse e tecido do cólon 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.

Isolamento e cultura primária de células de camundongo aórtica endoteliais

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.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e 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.

Isolamento, caracterização e alto Throughput análise de fluxo extracelular de Mouse primária Renal Tubular as células epiteliais

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.

Coleção celular única de células trophoblastos em embriões humanos de estágio de peri-implantação

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