Ce travail a été soutenu par les Instituts nationaux de la santé Grant 5KO8DK085136-02 à JY.

Une. Obtenir Colon tissus Souris Un protocole pour effectuer des expériences sur les animaux, en précisant les motifs et les objectifs de la recherche, a été soumis et approuvé par notre bureau institutionnel de la surveillance des animaux de recherche. <ol><li> Euthanize la souris avec de l&#39;isoflurane inhalation suivie par dislocation cervicale. </li><li> Faire une incision médiane ventrale, retirer l&#39;intestin grêle loin et identifier le côlon. Rétracter la vessie et de l&#39;utérus (si présent) latéralement et identifier l&#39;os pubien et le couper avec des ciseaux fins pour exposer le rectum. </li><li> Disséquer le mésentère dorsal du rectum hors du côlon et de mobiliser le côlon par l&#39;anus pour le caecum, et sectionner pour le débrancher du côlon de l&#39;intestin grêle. </li><li> Placer l&#39;échantillon dans de la glace-froid tampon phosphate salin (PBS). </li><li> Remplir une seringue de 10 ml avec du PBS glacé et vider le côlon lumière plusieurs fois pour effacer son contenu, puis couper le ope du côlonn utilisant la longueur sage-ciseaux. </li><li> Placer les deux points dans un tube conique de 50 ml contenant 20 ml de PBS glacé. Le côlon n&#39;a pas à être placée dans une orientation particulière quelconque. Laver le côlon par le remplacement du PBS jusqu&#39;à ce que le liquide est clair sans particules. </li></ol> Humain Un protocole d&#39;obtenir les tissus humains de patients en chirurgie, en précisant l&#39;importance de l&#39;obtention de ce tissu ainsi que l&#39;évaluation des risques et des avantages potentiels, a été soumis et approuvé par notre bureau institutionnel du Programme de Human Research Protection. <ol><li> collaboration de recherche avec un chirurgien colorectal est nécessaire d&#39;être en mesure d&#39;obtenir les tissus humains pour votre recherche. Un protocole pour obtenir des tissus du côlon humain doit être soumis et approuvé par votre comité d&#39;examen institutionnel. </li><li> Un consentement éclairé écrit est obtenu à partir du patient avant l&#39;intervention chirurgicale. </li><li> Le chirurgien va effectuer une résection du côlon dans le standmode ard. Une fois que le côlon a été retiré du patient, l&#39;échantillon est immédiatement retiré à la pathologie. 0,5 dans la section de pleine épaisseur de paroi du côlon n&#39;est pas nécessaire pour l&#39;évaluation pathologique est prévu et est immédiatement placé sur PBS glacé. Ce spécimen ne doit pas inclure le mésentère du côlon, et les appendices épiploïques doit être retiré aussi bien, puisque la présence de graisse aura une incidence sur le processus de digestion. </li></ol> 2. Dénuder cellules épithéliales <ol><li> Incuber le côlon de souris entière dans 25 ml de 5 mM d&#39;EDTA dans du HBSS (température ambiante) dans un tube conique de 50 ml. Placer le tube conique dans un bain d&#39;air de C en secouant à 37 ° (250 rpm) pendant 15 min. Après 15 min, soigneusement verser le liquide et remplir le tube avec 25 ml d&#39;EDTA 5 mM, répéter pour un total de 5 lavages. Pour côlon humain, après l&#39;obtention d&#39;une bande de ½ pouce de la paroi du côlon du pathologiste couper le côlon avec des ciseaux en quatre morceaux de taille égale. Placer deux des pièces en un seul tube de 50 ml conique, etplacer les deux morceaux restants de tissus du côlon dans 50 ml tube conique séparé. Puis incuber avec 25 ml d&#39;EDTA 5 mM dans du HBSS et effectuer les lavages, comme décrit ci-dessus. </li><li> Rincer deux fois le côlon dans 20 ml de PBS glacé. </li><li> Placer le tissu de côlon de souris dans un nouveau tube conique de 50 ml contenant 20 ml de RPMI-10, 5 U de dispase, et 2000 U de collagénase D (température ambiante). Pour les tissus du côlon humain, utiliser deux fois le montant de la dispase et de la collagénase (20 U dispase, 4000 U collagénase D). </li><li> Placer le tube dans un bain d&#39;air à 37 ° C en secouant orienté verticalement (S&#39;il est placé horizontalement, il ya trop d&#39;aération et des dommages aux cellules). Agiter à 250 tpm pour un maximum de 60 min. Après exposition à la dispase et de la collagénase D, le tissu du côlon va commencer à digérer et le tissu du côlon va commencer à regarder filandreuse. Cela prend habituellement environ 30 min. Lorsque le colon a cette apparence, enlever le côlon des enzymes. </li><li> Agiter chaque tube de haut en bas 2-4 fois pour briser le tissu. PEllet le tissu à 200 x g dans une centrifugeuse de paillasse (4 ° C) pendant 5 min. Versez délicatement le surnageant et le jeter. </li></ol> 3. Myofibroblastes Isolement et culture <ol><li> Remettre en suspension chaque culot par addition de 10 ml de tampon de lyse ACK (47 ° C). Centrifuger à 200 g pendant 5 min (4 ° C), verser et jetez le surnageant. </li><li> Remettre en suspension chaque culot par addition de 10 ml de RPMI-5 avec une pipette. </li><li> Passer la moitié de la suspension de cellules (5 ml) à travers un tamis de maille 70 um en utilisant une pipette de 10 ml dans une boîte de TC-traité 100 mm. Puis ajouter un supplément de 15 ml de RPMI-5. Répéter l&#39;opération avec les 5 ml restants de suspension cellulaire dans un plat de TC-traité 100 mm différente. Un colon de la souris va donc produire deux plats de TC traité 100 mm. Une bande de ½ pouce de tissu du côlon humain donnera un total de quatre plats de TC traité de 100 mm. </li><li> Placez les boîtes de culture de tissus dans un incubateur à CO 2 de 10% à 37 ° C. Les conditions de culture sont les mêmes fou de la souris et les cellules humaines primaires. </li><li> Après 3 heures, lavez délicatement les cellules non adhérentes avec deux changements de HBSS. Le flottement de débris doit être lavé délicatement. Les cellules adhérentes sont composées de cellules épithéliales, les macrophages et les myofibroblastes. Une semaine après l&#39;ensemencement, seulement myofibroblastes fracture, les macrophages et les cellules épithéliales sénescence après le premier passage. Ajouter frais RPMI-5 et cultiver les cellules dans la culture cellulaire. </li><li> Les cellules sont soumises à des passages par traitement à la trypsine pendant 5 minutes et sont divisées 2:1. </li></ol> Abréviations HBSS: solution saline équilibrée de Hank; RPMI-5: Roswell Park Memorial Institute médias; ACK: Ammonium-chlorure de potassium-; FCS: sérum de veau foetal, TC-traité: la culture de tissu traité Solutions Tampon de lyse ACK - (4,15 g de NH 4 Cl, 0,5 g de KHCO3, 18,6 mg Na 2 EDTA, 400 ml H 2 O, ajuster le pH à 7.2 à 7.4 avec HCl 1 N). Filtre stérilisablee à travers un filtre de 0,2 um et stocker à 4 ° C. RPMI-5 - (pour faire 500 ml: 454,5 ml de RPMI, 25 ml de FCS, 5 ml 200 mM de L-glutamine, 5 ml 1 M HEPES, pH 7,4, 5 ml pyruvate de sodium 100 mM, 5 ml 100x Pen-Strep, 500 ml 50 mM B-ME dans PBS).

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Les auteurs ont rien à révéler.

La technique générale est similaire pour l&#39;utilisation de la souris ou l&#39;autre ou du côlon humain. Cette technique peut également être utilisée pour isoler des myofibroblastes par l&#39;intestin grêle. Les détails spécifiques pour l&#39;isolement des myofibroblastes de 1. souris et 2. côlon humain sont discutés ci-dessous. Une. Colon souris L&#39;irrigation du colon de la souris est facilitée par la fixation d&#39;un 4 à 16 G Popper aiguille à ...

La régulation de la fonction gastro-intestinale (GI), implique des interactions complexes et dynamiques entre la couche de muqueuse et son mésenchyme sous-jacent. Ces interactions servent normalement à maintenir l&#39;homéostasie, mais peuvent également conduire au développement d&#39;états pathologiques dans des conditions pathologiques 1. Les myofibroblastes sont une sous-population de cellules du stroma sous-jacent qui se trouvent à la couche epitheliale qui communiquent dans un mode paracrine environnante avec des sous-populations de cellules pour réguler un certain nombre de processus critiques tractus gastro-intestinal comprenant la régénération de la muqueuse, la réparation, la fibrose et 2. La lignée cellulaire 18Co est un exemple d&#39;une lignée humaine du colon des myofibroblastes cellulaire qui a été largement utilisé pour étudier la fonction des myofibroblastes, car il partage un grand nombre des caractéristiques de primaire en myofibroblastes in situ. Il s&#39;agit notamment de l&#39;expression de la protéine d&#39;a-actine du muscle lisse (α-SMA) et la vimentine, ainsi que l&#39;expression d&#39;un certain nombredes récepteurs de surface cellulaire tels que le récepteur épidermique de facteur de croissance, ou des récepteurs pour 3,4 de l&#39;acide lysophosphatidique. En raison de ces caractéristiques ultrastructurales communes, ainsi que des similitudes dans la fonction biologique 5, la lignée cellulaire 18Co a été largement utilisée pour étudier la fonction des myofibroblastes dans le cadre de nombreux états pathologiques comme la maladie inflammatoire de l&#39;intestin ou 3,6 du cancer colorectal. Cependant, il ya des limites et des préoccupations inhérentes lors de l&#39;utilisation d&#39;une lignée cellulaire. Il s&#39;agit notamment de l&#39;instabilité génotypique au cours du temps qui se différencie des lignées cellulaires à partir de cellules primaires, en modifiant le phénotype et la fonction biologique de la cellule, y compris les taux et les interactions avec d&#39;autres populations cellulaires croissance. Les lignées cellulaires n&#39;ont pas non plus les composantes normales du micro-environnement de GI (épithéliales, stromales, et des composants vasculaires), ce qui est un obstacle majeur à leur utilisation. Par conséquent, les conclusions tirées de la recherche en ligne de cellule expérimentale nécessite une validation supplémentaire pour réduire la risk de mauvaise interprétation. Myofibroblastes primaires peuvent être obtenus à partir de tissus de côlon humain et la souris pour confirmer les résultats expérimentaux identifiés dans une lignée cellulaire 7. La méthode a été décrite à l&#39;origine par Mahida et al. Huit, et notre protocole est une des nombreuses techniques bien décrits qui peuvent être utilisés pour isoler des myofibroblastes primaires à partir de la souris et du côlon humain 9. Pour tout modèle de souris de la maladie du côlon, cette technique peut être utilisée pour isoler les myofibroblastes primaires pour étudier la façon dont ils interagissent avec les cellules voisines ou contribuer à la fois normal ou pathologique GI traite 10,11. Cette technique peut être utilisée pour étudier comment les myofibroblastes contribuent à la cicatrisation des muqueuses, de la fibrose, et le développement d&#39;adénomes et de carcinomes colorectaux. Myofibroblastes primaires peuvent également être obtenus à partir de tissu de colon humain qui a été réséqué au moment de l&#39;opération de bénigne (maladie inflammatoire de l&#39;intestin, les sténoses, la diverticulite) ou c malinonditions. Les cellules primaires isolées à partir de tissu de côlon humain et de souris peuvent être cultivées dans une culture de cellules et utilisées sur un nombre limité de passages.

Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Dispase I</td>
 <td>Invitrogen/GIBCO</td>
 <td>#17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche</td>
 <td>#1 088 858</td>
 <td></td>
 </tr>
 <tr>
 <td>4 in, 16 G Popper blunt-end needle</td>
 <td>Fisher</td>
 <td>#14-825-16K</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation of primary myofibroblasts from mouse and human colon tissue

Le myofibroblastes est une cellule stromale de l&#39;appareil gastro-intestinal (GI) qui a gagné une attention considérable de son rôle essentiel dans de nombreuses fonctions gastro-intestinales. Bien que plusieurs lignées cellulaires myofibroblastes sont disponibles dans le commerce pour étudier ces cellules in vitro, les résultats de la recherche provenant d&#39;une lignée de cellules exposées à des conditions de culture de cellules expérimentales ont des limites inhérentes dues à la nature trop réductrice de l&#39;œuvre. L&#39;utilisation de myofibroblastes primaires offre un grand avantage en termes de confirmer les résultats expérimentaux identifiés dans une lignée cellulaire. Isolement des myofibroblastes primaires à partir d&#39;un modèle animal pour l&#39;étude permet de myofibroblastes dans des conditions qui imitent plus près de l&#39;état pathologique en cours d&#39;étude. Isolation des myofibroblastes primaires de tissus du côlon humain fournit sans doute des données expérimentales les plus pertinentes, car les cellules viennent directement de patients atteints de la maladie sous-jacente. Nous décrivons une technique bien établie qui peut être utilized pour isoler myofibroblastes primaires à partir de la souris et le tissu du côlon humain. Ces cellules isolées ont été caractérisées pour être l&#39;alpha-actine musculaire lisse et la vimentine positif, et la desmine négatif, compatible avec les myofibroblastes sous-épithéliaux intestinaux. Cellules de myofibroblastes primaires peuvent être cultivées dans une culture cellulaire et utilisés à des fins expérimentales sur un nombre limité de passages.

Une fois isolées, les myofibroblastes humains primaires peuvent être cultivées dans une culture cellulaire et utilisés sur un nombre limité de passages (jusqu&#39;à passage 4). Ces cellules sont caractérisées comme étant un actine musculaire lisse et la vimentine positif et négatif desmine (figure 1A), compatible avec les myofibroblastes sous-épithéliales intestinales 5,7. Ils ont aussi une morphologie stellaire caractéristique (figure 1B). Myofibroblastes primai...

Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Le myofibroblastes est une cellule influent stromale du tractus gastro-intestinal qui régule les processus physiologiques importants dans les états normaux et pathologiques. Nous décrivons une technique qui permet l&#39;isolement des myofibroblastes primaires à partir de la souris et le tissu du côlon humain, qui peut être utilisé pour l&#39;expérimentation in vitro.

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-of-primary-myofibroblasts-from-mouse-and-human-colon-tissue

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Biology

19.5K vues.  UCLA.  Le myofibroblastes est une cellule influent stromale du tractus gastro-intestinal qui régule les processus physiologiques importants dans les états normaux et pathologiques. Nous décrivons une technique qui permet l'isolement des myofibroblastes primaires à partir de la souris et le tissu du côlon humain, qui peut être utilisé pour l'expérimentation in vitro. 

Vidéo: Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Isolement des noyaux neuronaux à partir de tissus post-mortem du cerveau de l&#39;homme

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Recherche

Biologie

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 ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve cellules souches adultes Isolation de cultures de fibroblastes humains primaires

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolement et analyse fonctionnelle des mitochondries de cellules cultivées et de tissus de souris

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Isolement de myofibroblastes de Mouse et œsophage humain

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

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 and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Isolement et caractérisation des fibroblastes cardiaques adultes et des myofibroblastes

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

Génération de monocouches épithéliales primaires murines du côlon à partir de cryptes intestinales

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolement et culture de kératinocytes oraux primaires du palais de souris adulte

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...