Les auteurs tiennent à remercier l&#39;équipe des services de création de l&#39;Université de Missouri Centre de soutien scolaire qui a produit cette vidéo. Ce travail a été soutenu par la Eunice Kennedy Shriver National Institute of Child Health &amp; Human Development de la National Institutes of Health, numéro d&#39;obtention HD055231

1. Dissection du placenta de souris <ol><li> Mettre en place paires d&#39;accouplement de souris. </li><li> Sur chacun des jours suivants, vérifiez la présence d&#39;un bouchon de copulation. Le jour où une prise est détecté est considéré comme 0,5 jours après le coït (dpc) 7. </li><li> Placentas peuvent être perçues au stade de la grossesse désirée, avec une dissection propre du placenta début possible avec le développement du placenta mature sur d 10.5. Ici, nous démontrons 14,5 dpc. Lors des volumes donnés, 40-60 placentas de 10,5 dpc, et 10-20 placentas sur 14,5 dpc permettra d&#39;obtenir une couche de trophoblaste clairement visible cellule. </li><li> Euthanasier la souris enceintes, et enlever l&#39;utérus, la coupe au niveau du col et le long de la mésomètre. </li><li> Avec des ciseaux pointus, couper entre chaque conceptus (figure 1A). </li><li> L&#39;utilisation de deux paires de pinces, saisir la paroi utérine à l&#39;arête de coupe, et tirer, enlever le myomètre (figure 1B)<html> . Une paire dentelée, pince modérément fines (courbe ou droite) plus une paire pinces très fines (# 5, par exemple) est recommandée. </li><li> En face du placenta, ouvrez la caduque avec des pinces ou des ciseaux, ce qui expose le placenta. Saisir le tissu utérin avec une paire pince, faites glisser une autre pince sous le placenta se séparer (figure 1C). Si nécessaire, le fœtus séparé et membranes fœtales à partir du placenta. Il n&#39;est pas possible d&#39;enlever tous les tissus déciduale par dissection sans perdre également le tissu placentaire, mais d&#39;autres tissus déciduale (pâle) peut être enlevé de la surface du placenta par un léger gommage avec une pince. Nous nous appuierons sur le gradient de Percoll une purification supplémentaire. </li><li> Placez chaque placenta disséqué dans 25 ml de glace froide tampon de dissociation dans un tube conique de 50 ml jusqu&#39;à ce que toutes les dissections sont terminées. Aux stades ultérieurs, émincer grossièrement tissu placentaire avec des ciseaux ou une lame de rasoir avant de les transférer dans la mémoire tampon de dissociation. </li></ol>tle &quot;&gt; 2. Isolement des cellules trophoblastiques <ol><li> Tube à bouchon conique sans serrer et placer dans un bain marie à 37 ° C pendant 45-60 minutes. Mélanger la solution de dissociation vigoureusement à la pipette environ toutes les 10 min. Le temps de digestion totale doivent être étroitement surveillés et il faudra probablement quelques essais et erreurs pour parvenir au recouvrement optimal des cellules. Les tissus doivent être en mesure de passer à travers une pipette de 10 ml, mais des morceaux visibles de tissu restera. Digestion excessive entraînera dans la survie cellulaire pauvres et l&#39;efficacité de placage. La survie des cellules et une efficacité optimales plaquage se produit lorsque des amas de quelques cellules sont présents, plutôt que lorsque la dissociation complète est réalisée. A l&#39;inverse, underdigestion mènera à la pureté réduite après la séparation percoll. Lorsque le point de digestion a été atteint, tube de place sur la glace pour arrêter l&#39;activité de la collagénase. </li><li> Faire passer la solution à travers un tamis cellulaire pour éliminer les matières non digérées, et centrifuger à 500 xg pendant 5 minutes. </li><li> Pour laver, retirercellules surnageant et remettre en suspension dans 10 ml de solution de lavage. Centrifuger à 500 xg pendant 5 minutes. </li><li> Lors de la centrifugation, préparer la solution percoll en mélangeant 9,6 ml percoll, 13,4 ml de solution de lavage et de 1,1 ml de milieu 10x 199 dans un tube de 50 ml appropriée pour centrifugation à grande vitesse. </li><li> Retirez la cellule surnageant et resuspendre le culot dans 2 ml de solution de lavage. </li><li> Ajouter à cellules Percoll solution et centrifuger @ 30000 xg pendant 30 minutes à 4 ° C. </li><li> Retirer le tube de centrifugeuse sans déranger le gradient. Pour visualiser le tube de gradient lieu, devant une source de lumière vive. Repérez la bande trophoblaste (figure 2), et aspirer avec une pipette, en prenant soin de ne pas déranger la bande de globules rouges ci-dessous. Volume de la bande trophoblaste sera 5-8 ml. </li><li> Transfert cellules trophoblastiques dans un tube conique de 50 ml. Ajouter la solution de lavage pour un volume total de 45 ml. </li><li> Centrifuger à 500 xg pendant 5 minutes. Retirer le surnageant et remettre en suspension dans la culture médium (NCTC-135). Les cellules peuvent être cultivées sur couche mince ou en plastique matrigel culture de tissus à 37 ° C et 5% de CO 2. Au jour 10.5, chaque placenta donne environ 50.000 cellules vivantes, dont 80-90% sont cytokératine positive. </li></ol> 3. Essai invasion trophoblastique <ol><li> Ajouter 1-2 ml du milieu de culture (NCTC-135) à chaque chambre matrigel et incuber à 37 °, 5% de CO 2 pendant 30-90 min. </li><li> Retirer moyen et ajouter 1,5 ml x 10 5 cellules fraîchement isolées en milieu 2ml à la chambre supérieure. Ajouter 1 ml de milieu de la chambre basse pour couvrir la surface inférieure membrane. </li><li> Cellules en culture dans des inserts pendant 18-24 heures. </li><li> Gratter les cellules et matrigel de l&#39;insert à l&#39;aide d&#39;un écouvillon en mousse. </li><li> Rincer le haut et le bas de la chambre à 2 ml de PBS. </li><li> Fixer pendant 5 min dans 1 ml de paraformaldéhyde 4% dans du PBS. </li><li> Colorer en remplissant la chambre basse avec 400 pl DAPI (300 nm) et incuber pendant 30 min. </li><li> Laver trois fois brièvement dans du PBS. </li><li&gt; Retirer les membranes de plaquettes avec une lame de scalpel tranchant. </li><li> Ajouter une chute de 10 pi de milieu de montage (glycérol à 50% dans du PBS) à glisser. En utilisant des pinces, poser la membrane sur la chute. Ajouter une autre baisse de 10 pi de milieu, puis lamelle avec soin. </li><li> Cellules et le nombre photographie. Veillez à orienter correctement la membrane (cellule vers le haut ou de la cellule vers le bas) pour le microscope utilisé. Image J logiciels (National Institutes of Health) comporte une fonction de comptage manuel qui peut être utilisé à cette fin, tout comme Adobe Photoshop ou un compteur manuel. </li></ol> 4. Les résultats représentatifs <img src="/files/ftp_upload/3202/3202fig1.jpg" alt="Figure 1" /> Figure 1. Dissection du placenta de souris. (A) L&#39;utérus est enlevé par une coupe suivant les oviductes (jaune), le long de la surface mesometrial, et au col de l&#39;utérus (partie inférieure). Conceptus individuels peuvent ensuite être séparés en coupant avec des ciseaux comme indiqué. (B) Le utérine<html> paroi est éliminé en plaçant deux pinces à la surface de coupe de l&#39;utérus, et à la déchirure le long de la face anti-mesometrial, à proximité du foetus et à distance du placenta. (C) Le placenta est ensuite isolé par préhension avec une pince coulissante et une autre entre le placenta et le tissu utérin. Le cordon ombilical et les membranes extra-embryonnaires d&#39;autres peuvent ensuite être séparés du placenta. <img src="/files/ftp_upload/3202/3202fig2.jpg" alt="Figure 2" /> Figure 2. Percoll séparation des cellules trophoblastiques. Après centrifugation, trois bandes majeures sera visible comme indiqué dans le dessin animé à gauche, et la photo à droite. La bande supérieure contient des débris cellulaires et des fibroblastes et sera le plus apparent. Une serré (rouge) près de la bande de fond contenant des globules rouges sera visible juste en dessous de la bande trophoblaste diffus. Agrégats cellulaires seront probablement vu dans la bande de trophoblaste, comme dans les deux montré juste à gauche de la flèche sur la photo. ove_content &quot;&gt; <img src="/files/ftp_upload/3202/3202fig3.jpg" alt="Figure 3" /> La figure 3. Cellules trophoblastiques invasives (flèche blanche) passe à travers la couche de Matrigel et les pores de la membrane, apparaissant sur ​​la surface inférieure de la membrane. Les pores sont également visibles (flèche noire). Le nombre de cellules sur la surface de la membrane entière ou une zone échantillon représentatif peut être compté. Une image en niveaux de gris de fluorescence DAPI nucléaire a été inversé pour améliorer la visualisation des cellules. <img src="/files/ftp_upload/3202/3202fig4.jpg" alt="Figure 4" /> Cellules trophoblastiques Figure 4. Peut être identifiée, et la pureté de la population isolée évalué par immunocoloration pour la cytokératine 7 (centre, magenta). Les cellules sont DAPI (en haut, bleu). Fusionnée image de fond.

<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>

Nous n&#39;avons rien à déclarer.

Dans cette vidéo, nous démontrons l&#39;isolement de cellules trophoblastiques du placenta de souris. Nous montrons ensuite un test in vitro pour l&#39;invasion du trophoblaste. Grâce à cette procédure, une grande partie, mais pas entièrement, la population pure de cellules trophoblastiques peuvent être obtenues. Si la pureté supplémentaire est nécessaire, de séparation du talon magnétique ou cytométrie en flux peut être réalisée, comme cela a été décrit pour les cellules trophoblastiques primaires de...

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

Le placenta est responsable du transport des nutriments, des gaz et des facteurs de croissance pour le fœtus, ainsi que l&#39;élimination des déchets. Ainsi, des défauts dans le développement placentaire avoir des conséquences importantes pour le fœtus et la mère, et sont une cause majeure de la létalité embryonnaire. Le type cellulaire majeur de la portion fœtale du placenta est le trophoblaste. Des cellules primaires de souris trophoblastes placentaires sont un outil utile pour étudier le développement normal et anormal du placenta, et contrairement à des lignées cellulaires, peuvent être isolés et utilisés pour étudier trophoblaste à des étapes spécifiques de la grossesse. En outre, des cultures primaires de trophoblaste de souris transgéniques peuvent être utilisées pour étudier le rôle des gènes particuliers dans les cellules placentaires. Le protocole présenté ici est basée sur la description de Thordarson et al. 1, dans lequel un gradient de Percoll est utilisé pour obtenir une population de cellules du trophoblaste relativement pur de placenta de souris isolées. Il est semblable à l&#39;utiliser plus largementméthodes d pour 2-3 trophoblaste humain isolement cellulaire. Pureté peut être évaluée par coloration immunocytochimique des cellules isolées pour la cytokératine 7 4. Ici, les cellules isolées sont ensuite analysés en utilisant un test invasion matrigel trophoblaste invasif pour évaluer in vitro 5-6. Les cellules envahies sont analysés par immunocytochimie et colorées pour le comptage.

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

Dans ce protocole, nous décrivons la dissection du placenta de la souris sur la grossesse D10.5, suivie de l&#39;isolement des cellules trophoblastiques en utilisant un gradient de Percoll. Nous avons ensuite démontrer l&#39;utilisation des cellules isolées dans un essai invasion matrigel.

Isolement des cellules trophoblastiques primaires de souris et le test invasion trophoblastique

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

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23.4K vues.  University of Missouri. Dans ce protocole, nous décrivons la dissection du placenta de la souris sur la grossesse D10.5, suivie de l'isolement des cellules trophoblastiques en utilisant un gradient de Percoll. Nous avons ensuite démontrer l'utilisation des cellules isolées dans un essai invasion matrigel.

Vidéo: 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.

Dérivation de cellules souches de la souris Trophoblast à partir de blastocystes

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

Recherche

Biologie

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%.

L&#39;isolement et la culture primaire de cellules de foie de rat

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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.

Isolement des cellules immunitaires des tumeurs primitives

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.

Isolement et culture de cellules primaires de souris acineuses du pancréas

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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.

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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.

Isolation des myofibroblastes primaires de souris et de tissus du côlon humain

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.

L&#39;isolement et la culture primaire de cellules endothéliales aortiques de souris

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.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

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.

Analyse de Flux extracellulaire isolement, caractérisation et un débit élevé de souris primaire tubulaire les cellules épithéliales rénales

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

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Isolement et culture des cellules endothéliales aortiques primaires des porcs miniatures

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...