Diese Arbeit wurde vom National Institutes of Health Grants 5KO8DK085136-02 bis JY unterstützt.

1. Erhalten Colon Tissue Maus Ein Protokoll zum Tierversuch durchzuführen, detailliert die Gründe und Ziele der Forschung, vorgelegt und von unserem institutionellen Amt für Tierforschung Aufsicht genehmigt wurde. <ol><li> Euthanize die Maus mit inhalativen Isofluran durch Genickbruch folgte. </li><li> Machen Sie einen ventralen Mittellinienschnitt, Einfahren der Dünndarm entfernt und Identifizierung der Doppelpunkt. Fahren Sie die Blase und Gebärmutter (falls vorhanden), die seitlich und Identifizierung des Schambeins und schneiden Sie es mit einer feinen Schere, um den Mastdarm aus. </li><li> Präparieren Sie die dorsalen Mesenterium des Mastdarms aus der Dickdarm-und Dickdarm zu mobilisieren, die aus dem After, um den Blinddarm, und es durchschneiden, um den Darm aus dem Dünndarm zu trennen. </li><li> Man gibt die Probe in eiskalter phosphatgepufferter Kochsalzlösung (PBS). </li><li> Füllen Sie eine Spritze mit 10 ml eiskaltem PBS und spülen den Darm Lumen mehrmals, um den Inhalt zu löschen, dann schneiden Sie den Doppelpunkt open der Länge nach mit einer Schere. </li><li> Setzen Sie den Doppelpunkt in einem 50 ml konischen Röhrchen mit 20 ml eiskaltem PBS. Der Darm nicht in einer bestimmten Ausrichtung angeordnet werden. Waschen Sie den Doppelpunkt durch den Austausch des PBS, bis die Flüssigkeit klar ohne Feinstaub. </li></ol> Mensch Ein Protokoll für die menschliche Gewebe von chirurgischen Patienten zu erhalten, detailliert die Bedeutung der Erhalt dieser Gewebe sowie eine Bewertung der potenziellen Risiken und Vorteile, vorgelegt und von unserem institutionellen Amt des Human Research Protection Program genehmigt wurde. <ol><li> Forschungskooperation mit einem kolorektalen Chirurgen notwendig ist, um in der Lage, menschliches Gewebe für Ihre Forschung zu erhalten. Ein Protokoll der menschlichen Dickdarmgewebe zu erhalten, sollte eingereicht werden und die Ihr Institutional Review Board genehmigt werden. </li><li> Schriftliche Einverständniserklärung von dem Patienten vor dem chirurgischen Eingriff erhalten. </li><li> Der Chirurg wird einen Doppelpunkt Resektion im Stand durchführenard Mode. Sobald der Darm aus dem Patienten entfernt worden ist, wird die Probe sofort entnommen Pathologie. Eine 0,5 in voller Dicke Abschnitt Darmwand nicht für die pathologische Auswertung benötigt wird und wird unmittelbar auf eiskaltem PBS gelegt. Dieses Exemplar sollte nicht die Dickdarm-Mesenteriums und epiploicum Anhänge sollten ebenfalls entfernt werden, da das Vorhandensein von Fett wird die Verdauung beeinflussen. </li></ol> 2. Entblößen Epithelzellen <ol><li> Inkubieren der gesamten Maus Kolon in 25 ml 5 mM EDTA in HBSS (Raumtemperatur) in einem 50 ml konischen Röhrchen. Legen Sie die konische Röhrchen in einem 37 ° C Luft-Bad Schütteln (250 rpm) für 15 min. Nach 15 Minuten vorsichtig abgießen der Flüssigkeit und füllen Sie den Schlauch mit 25 ml 5 mM EDTA, wiederholen Sie für insgesamt 5 Wäschen. Für menschliche Dickdarm, nach Erhalt einer ½ Zoll-Streifen der Darmwand von der Pathologe schneiden den Doppelpunkt mit einer Schere in vier gleich große Stücke schneiden. Legen zwei der Stücke in einen 50 ml konischen Röhrchen undDie restlichen zwei Stücke von Dickdarm-Gewebe in einem separaten 50 ml konischen Röhrchen. Dann inkubiert mit 25 ml 5 mM EDTA in HBSS und führen Waschungen wie oben beschrieben. </li><li> Zweimal Spülen Sie den Dickdarm in 20 ml eiskaltem PBS. </li><li> Die Maus Dickdarmgewebe in eine neue 50 ml konischen Röhrchen, enthaltend 20 ml RPMI-5, 10 U Dispase und 2.000 U Kollagenase D (Raumtemperatur). Für den menschlichen Dickdarmgewebe, verwenden Sie die doppelte Menge an Dispase und Kollagenase (20 U Dispase, 4.000 U Kollagenase D). </li><li> Das Röhrchen wird in einem 37 ° C schütteln Luftbad vertikal ausgerichtet (Wenn horizontal platziert, es gibt zu viel Be-und Zellschäden). Schütteln bei 250 rpm für bis zu 60 min. Nach Kontakt mit dem Dispase und Collagenase D, wird der Dickdarmgewebe zu starten, um zu verdauen und die Dickdarmgewebe beginnt zu sehnig aussehen. Dies dauert in der Regel ca. 30 min. Wenn der Darm hat diese Erscheinung, entfernen den Dickdarm von den Enzymen. </li><li> Schütteln Sie jedes Rohr nach oben und unten 2-4 mal zu brechen das Gewebe. PEllet das Gewebe bei 200 × g in einer Tischzentrifuge (4 º C) für 5 min. Vorsichtig gießen Sie den Überstand und entsorgen. </li></ol> 3. Myofibroblasten Isolierung und Kultur <ol><li> Resuspendieren jedes Pellet durch Zugabe von 10 ml ACK-Lysepuffer (47 ° C). Zentrifuge wieder bei 200 g für 5 min (4 ° C), abgießen und den Überstand verwerfen. </li><li> Resuspendieren jedes Pellets durch Zugabe von 10 ml RPMI-5 mit einer Pipette. </li><li> Geben Hälfte der Zellsuspension (5 ml) über einen 70 &amp;mgr; m-Mesh-Sieb mit einer 10 ml Pipette in ein 100 mm TC-behandelten Teller. Dann fügen Sie weitere 15 ml RPMI-5. Wiederholen mit den verbleibenden 5 ml der Zellsuspension in einen anderen 100 mm TC-behandelten Teller. Eine Maus Doppelpunkt wird daher ergeben zwei 100 mm TC-behandelt Gerichte. Eine ½ Zoll-Streifen der menschlichen Dickdarmgewebe wird insgesamt vier 100-mm-TC-behandelt Gerichte ergeben. </li><li> Platzieren der Gewebekulturplatten in einer 10% CO 2-Inkubator bei 37 ° C Die Kulturbedingungen sind die gleichen foder Maus und humane primäre Zellen. </li><li> Nach 3 Std., sanft abwaschen nicht-adhärenten Zellen mit zwei Änderungen HBSS. Die Trümmer Floating sollte vorsichtig ausgewaschen werden. Adhärente Zellen epithelialen Zellen, Makrophagen und Myofibroblasten besteht. Eine Woche nach der Aussaat, Myofibroblasten nur teilen, Makrophagen und Epithelzellen altern nach dem ersten Durchgang. In frischem RPMI-5 und wachsen die Zellen in Zellkultur. </li><li> Die Zellen werden durch Trypsinierung für 5 min agiert und sind gespalten 02.01. </li></ol> Abkürzungen HBSS: Hank-Salzlösung; RPMI-5: Roswell Park Memorial Institute Medien; ACK: Ammonium-Chlorid-Kalium; FCS: fetales Kälberserum, TC-behandelt: Gewebekultur behandelt Lösungen ACK-Lysepuffer - (4,15 g NH 4 Cl, 0,5 g KHCO 3, 18,6 mg Na 2 EDTA, 400 ml H 2 O, pH-Wert auf 7,2-7,4 mit 1 N HCl). Filter sterilisierbare durch einen 0,2 um Filter und bei 4 ° C RPMI-5 - (bis 500 ml machen: 454,5 ml RPMI, 25 ml FCS, 5 ml 200 mM L-Glutamin, 5 ml 1 M HEPES, pH 7,4, 5 ml 100 mM Natrium-Pyruvat, 5 ml 100x Pen-Strep, 500 ml 50 mM B-ME in PBS).

<ol>
	<li>Armaka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cell+targeting+by+TNF+as+a+common+pathogenic+principle+in+chronic+inflammatory+joint+and+intestinal+diseases.">Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases.</a> J. Exp. Med. 205, 331-337 (2008).</li><li>Powell, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+cells+important+in+health+and+disease.">Paracrine cells important in health and disease.</a> Am. J. Physiol. 277, C1-C9 (1999).</li><li>Yoo, J., Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D1+mediates+synergistic+MMP-3+expression+induced+by+TNF-alpha+and+bradykinin+in+human+colonic+myofibroblasts.">Protein kinase D1 mediates synergistic MMP-3 expression induced by TNF-alpha and bradykinin in human colonic myofibroblasts.</a> Biochem. Biophys. Res. Commun. 413, 30-35 (2011).</li><li>Yoo, J., Chung, C., Slice, L., Sinnett-Smith, J., Rozengurt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+D+mediates+synergistic+expression+of+COX-2+induced+by+TNF-{alpha}+and+bradykinin+in+human+colonic+myofibroblasts.">Protein kinase D mediates synergistic expression of COX-2 induced by TNF-{alpha} and bradykinin in human colonic myofibroblasts.</a> Am. J. Physiol. Cell Physiol. 297, C1576-C1587 (2009).</li><li>Valentich, J. D., Popov, V., Saada, J. I., Powell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+an+intestinal+subepithelial+myofibroblast+cell+line.">Phenotypic characterization of an intestinal subepithelial myofibroblast cell line.</a> Am. J. Physiol. 272, C1513-C1524 (1997).</li><li>Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E., Yoo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+potentiates+lysophosphatidic+acid-induced+COX-2+expression+via+PKD+in+human+colonic+myofibroblasts.">TNF-alpha potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 300, G637-G646 (2011).</li><li>Yoo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+induces+upregulation+of+EGFR+expression+and+signaling+in+human+colonic+myofibroblasts.">TNF-alpha induces upregulation of EGFR expression and signaling in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 302, G805-G814 (2012).</li><li>Mahida, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+colonic+subepithelial+myofibroblasts+express+extracellular+matrix+proteins+and+cyclooxygenase-1+and+-2.">Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2.</a> Am. J. Physiol. 273, G1341-G1348 (1997).</li><li>De Wever, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+and+potentiation+of+DNA+damage+response+by+fibronectin+in+human+colon+cancer+cells+and+tumor-derived+myofibroblasts.">Priming and potentiation of DNA damage response by fibronectin in human colon cancer cells and tumor-derived myofibroblasts.</a> Int. J. Oncol. 39, 393-400 (2011).</li><li>Edwards, R. A., Smock, A. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+arachidonate+release+and+PGE2+production+in+Gi+alpha2-deficient+intestinal+and+colonic+subepithelial+myofibroblasts.">Defective arachidonate release and PGE2 production in Gi alpha2-deficient intestinal and colonic subepithelial myofibroblasts.</a> Inflamm. Bowel Dis. 12, 153-165 (2006).</li><li>Hoang, B., Trinh, A., Birnbaumer, L., Edwards, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+MAPK-+and+PGE2-dependent+IL-11+production+in+Gialpha2-/-+colonic+myofibroblasts.">Decreased MAPK- and PGE2-dependent IL-11 production in Gialpha2-/- colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1511-G1519 (2007).</li><li>Li, Z. B., Kollias, H. D., Wagner, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myostatin+directly+regulates+skeletal+muscle+fibrosis.">Myostatin directly regulates skeletal muscle fibrosis.</a> J. Biol. Chem. 283, 19371-19378 (2008).</li><li>Saada, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subepithelial+myofibroblasts+are+novel+nonprofessional+APCs+in+the+human+colonic+mucosa.">Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa.</a> J. Immunol. 177, 5968-5979 (2006).</li><li>Lahar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+subepithelial+myofibroblasts+support+in+vitro+and+in+vivo+growth+of+human+small+intestinal+epithelium.">Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium.</a> PLoS One. 6, e26898 (2011).</li></ol>

Autoren haben nichts zu offenbaren.

Die allgemeine Technik ist ähnlich wie bei der Verwendung von entweder Maus-oder menschlichen Kolon. Diese Technik kann auch verwendet werden, um Myofibroblasten aus dem Dünndarm zu isolieren. Konkrete Angaben zur Isolierung von Myofibroblasten aus 1. Maus und 2. menschlichen Dickdarm werden nachstehend erörtert. 1. Maus Colon Spülung des Dickdarms Maus wird durch Anbringen eines 4 in 16 g Popper stumpfendig Nadel an einem Ende des Dickdarms erleichtert. Dies hi...

Die Regelung der Funktionstrakt gastrointestinale (GI) ist mit komplexen, dynamischen Wechselwirkungen zwischen der Schleimhautschicht und der zugrunde liegenden Mesenchym. Diese Interaktionen dienen in der Regel, um die Homöostase aufrecht zu erhalten, kann aber auch auf die Entwicklung von Krankheitszuständen führen, unter pathologischen Bedingungen ein. Myofibroblasten sind eine Subpopulation von Stromazellen an der Epithelschicht, die in einer Umgebung mit parakrin Zell-Subpopulationen, um eine Reihe von kritischen Prozessen, die GI-Trakt Schleimhautregeneration, Reparatur und Fibrose 2 einschließen, regeln, welcher unterhalb kommunizieren. Die 18Co-Zelllinie ist ein Beispiel eines menschlichen Kolon-Myofibroblasten Zelllinie, die weit verbreitet ist, um Myofibroblasten Funktion zu studieren, weil es viele der Eigenschaften von Primär in situ Myofibroblasten Aktien. Dazu gehören die Proteinexpression von einer glatten Muskel-Aktin (α-SMA) und Vimentin, als auch die Expression einer ReiheZelloberflächenrezeptoren wie der epidermale Wachstumsfaktor-Rezeptor oder Rezeptoren für Lysophosphatidsäure 3,4. Da dieser gemeinsamen Merkmale ultra sowie Ähnlichkeiten in biologischen Funktion 5 hat die 18Co-Zelllinie wurde ausgiebig verwendet, um Myofibroblasten Funktion im Zusammenhang mit vielen Krankheitszuständen, wie entzündliche Darmerkrankung oder Darmkrebs 3,6 studieren. Allerdings gibt es inhärente Beschränkungen und Bedenken bei der Verwendung einer Zelllinie. Dazu gehören genotypische Instabilität über die Zeit, die Zelllinien aus Primärzellen unterscheidet, Änderung der Phänotyp und die biologische Funktion der Zelle, einschließlich Wachstumsraten und Wechselwirkungen mit anderen Zellpopulationen. Zellinien fehlt auch die normalen Komponenten der GI-Mikroumgebung (Epithel-, Stroma-und Gefäß Komponenten), die eine große Einschränkung ihrer Verwendung ist. Daher sind die Schlussfolgerungen aus experimentellen Zelllinie gezogen Forschung erfordert eine weitere Bestätigung, um die ri reduzierensk von Fehlinterpretationen. Primäre Myofibroblasten aus Mensch und Maus Dickdarmgewebe erhalten werden, um in einer Zelllinie 7 identifiziert experimentellen Ergebnisse zu bestätigen. Das Verfahren wurde ursprünglich von Mahida, et al. 8 beschrieben, und unser Protokoll ist eine von vielen gut beschriebene Techniken, die verwendet werden kann, um primäre Myofibroblasten aus Maus und Mensch Colon 9 isolieren. Für jede Maus-Modell der Dickdarmerkrankung, kann diese Technik verwendet, um primäre Myofibroblasten zu isolieren, um zu studieren, wie sie mit Nachbarzellen interagieren, oder dazu beitragen, sowohl normalen oder pathologischen GI 10,11 verarbeitet werden. Diese Technik kann verwendet werden, um zu untersuchen, wie Myofibroblasten tragen zur Heilung, Fibrose, und die Entwicklung von kolorektalen Adenomen und Karzinomen der Schleimhaut werden. Primär Myofibroblasten auch von humanen Kolon-Gewebe, das bei der Operation für gutartige (entzündliche Darmerkrankung, Strikturen, Divertikulitis) oder bösartig c reseziert worden ist, erhalten werden,ie Bedingungen. Isolierten primären Zellen aus Mensch und Maus Dickdarmgewebe in Zellkultur gezüchtet und über eine begrenzte Anzahl von Durchgängen verwendet werden.

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

Die Myofibroblasten eine Stromazellen des Magen-Darm-Trakt (GI), die gewonnen hat beträchtliche Aufmerksamkeit für seine kritische Rolle in vielen Funktionen GI. Während mehrere Myofibroblasten Zelllinien sind im Handel erhältlich, diese Zellen in vitro zu untersuchen, Forschungsergebnisse aus einer Zelllinie, experimentelle Zellkulturbedingungen ausgesetzt haben inhärente Beschränkungen aufgrund der übermäßig reduktionistischen Art der Arbeit. Einsatz von Primär Myofibroblasten bietet einen großen Vorteil in Bezug auf die Bestätigung in einer Zelllinie identifiziert experimentellen Befunde. Isolierung von primären Myofibroblasten aus einem Tiermodell erlaubt die Untersuchung von Myofibroblasten unter Bedingungen, die stärker nachzuahmen der Krankheitszustand untersucht. Isolation von Primär-Myofibroblasten aus menschlichen Dickdarmgewebe bietet wohl die wichtigsten experimentellen Daten, da die Zellen kommen direkt von Patienten mit der Grunderkrankung. Wir beschreiben eine etablierte Technik, die u seintilized Primär Myofibroblasten sowohl aus Maus und Mensch Dickdarmgewebe zu isolieren. Diese isolierten Zellen wurden charakterisiert, um alpha-smooth muscle Aktin und Vimentin-positiven und-negativen Desmin, im Einklang mit subepithelialen Myofibroblasten Darm sein. Primär Myofibroblasten Zellen in Zellkultur gezüchtet und für experimentelle Zwecke über eine begrenzte Anzahl von Durchgängen verwendet werden.

Einmal isoliert, primäre menschliche Myofibroblasten in Zellkultur gezüchtet und über eine begrenzte Anzahl von Durchgängen verwendet werden (bis zu Passage 4). Diese Zellen werden als ein-smooth muscle Aktin und Vimentin positiv gekennzeichnet, und Desmin-negativ (Abbildung 1A), im Einklang mit Darm subepithelialen Myofibroblasten 5,7. Sie haben auch eine charakteristische stern Morphologie (Fig. 1B). Primär Myofibroblasten kann dann verwendet werden, um eine Zelllinie ...

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

Die Myofibroblasten ist ein einfluss Stromazellen des Magen-Darm-Trakt, die wichtige physiologische Prozesse in normalen und Krankheitszustände regelt. Wir beschreiben eine Technik, die für die Isolierung von primären Myofibroblasten aus Maus und menschlichen Dickdarmgewebe, das für die in vitro Experimente verwendet werden können, ermöglicht.

Isolierung von Primär Myofibroblasten aus Maus und Mensch 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 ...

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

Research

JoVE Journal

Biology

19.5K Aufrufe.  UCLA.  Die Myofibroblasten ist ein einfluss Stromazellen des Magen-Darm-Trakt, die wichtige physiologische Prozesse in normalen und Krankheitszustände regelt. Wir beschreiben eine Technik, die für die Isolierung von primären Myofibroblasten aus Maus und menschlichen Dickdarmgewebe, das für die in vitro Experimente verwendet werden können, ermöglicht. 

Serie: 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.

Neuronale Kerne Isolation von Human Postmortem Gehirngewebe

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

Forschung

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.

Isolierung von Immunzellen aus Primärtumoren

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&#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.

Naive Adult Stem Cells Isolation von primären humanen Fibroblasten Kulturen

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.

Isolierung und funktionale Analyse von Mitochondrien aus kultivierten Zellen und Maus-Gewebe

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.

Isolierung von Myofibroblasten aus Maus und Mensch Ösophagus

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.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

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.

Isolierung und Kultur der primären Maus Keratinozyten aus neonatalen und erwachsenen Maus Haut

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.

Isolierung und Charakterisierung von adulten Herz-Fibroblasten und Myofibroblasten

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.

Erzeugung von murinen primären Dickdarmepithel-Monoschichten aus Darmkrypten

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.

Isolierung und Kultur von primären oralen Keratinozyten aus dem erwachsenen Mausgaumen

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