Diese Forschung wurde unterstützt durch Zuschüsse aus der Israel Science Foundation (Nr. 1418/11) an und AHH (Nr. 1933-1912) zu AW, der Deutsch-Israelischen Stiftung für junge Forscher (GIF Young) zu AHH, und die Dr. I unterstützt . Cabakoff Forschung Endowment Fund an der Hebrew University-Hadassah School of Dental Medicine, um AHH und AW.

Bereiten Sie im Voraus: <ul><li> PBS + 2% FCS </li><li> PBS + 2% FCS mit 2 mg / ml Kollagenase Typ II und 1 mg / ml DNAse Typ I (1 ml pro Probe) </li><li> Sterilisiert chirurgische Instrumente </li><li> 0,5 M EDTA-Lösung </li></ul> 1. Obere Gingival Exzision Technik <ol><li> Euthanize Mäusen mit zugelassenen IACUC Führung. </li><li> Schneiden Sie beide Seiten der Mundhöhle, einschließlich der Wangen und des Unterkiefers Ramus mit einem spitz / stumpf gerade Schere. </li><li> Ziehen Sie den Unterkiefer. </li><li> Schneiden Sie die weichen und harten Gewebe in einer gedachten Linie 1 mm hinter den Backenzähnen Drittel mit Standard-Schere. Richten Sie die Schere senkrecht zur Ebene des palatinalen Knochen (2A, blaue Linien). </li><li> Inzision der weichen und harten Gewebe 2 mm hinter den Schneidezähnen (2A, blaue Linien). </li><li> Ziehen Sie den vorderen Teil des Mundes. Die Nasenhöhle ist nach diesem Schritt beobachtet werden. </li><li> Legen Sie die Schereparallel zum Gaumen, legte ein Messer in die Nasenhöhle, sollte die andere Klinge in der Vorhalle einzuschneiden. Schneiden bis zum hinteren Einschnitt (2A, grüne Linien). Wiederholen Sie diesen Schritt auf beiden Seiten des Oberkiefers. Am Ende der Oberkiefer ist vom Rest des Schädels (2B) löst. </li><li> Entfernen Sie den Oberkiefer, schneiden Sie es in der Mitte Naht (Abbildung 2, schwarz gestrichelte Linie) und schneiden Sie die palatinale Gewebe jedes Hemi-Oberkiefer bis zum Erreichen der Alveolarknochen. </li><li> Schälen Sie die gingivalen Gewebe (sowohl Hemi-Oberkiefer) von ihren vorderen Rand mit Adson Pinzette (ohne Zähne). </li><li> Legen Sie das ausgeschnittene Zahnfleisch in einem Teller mit PBS + 2% FCS. </li></ol> 2. Gingival Verarbeitung <ol><li> Setzen Sie den ausgeschnittenen Zahnfleisch in einem Gewebe Kulturschale (35 x 10 mm) mit 1 ml PBS + 2% FCS, 2 mg / ml Collagenase Typ II und 1 mg / ml DNAse Typ I. </li><li> Mince gut das Gewebe mit einem N ° 15 sterile Klinge. </li><li> Transfer das Gewebe von der Platte auf einem 15 ml konischen Röhrchen. </li><li> Waschen Sie die Gewebe / Zellen noch auf der Platte mit 1 ml PBS + 2% FCS und Transfer zum gleichen Reagenzglas. </li><li> Inkubation in einem Schüttler Inkubator für 20 min bei 37 ° C, 200 rpm (Empfehlung: Heizen Sie den Inkubator). </li><li> In 20 ul 0,5 M EDTA und Inkubation für weitere 10 min bei 37 ° C, 200 UpM. </li><li> Füllen Sie das Teströhrchen bis zu 12 ml mit PBS + 2% FCS und Zentrifuge bei 4 ° C, 400 xg für 8 min (Empfehlung: pre-chill die Zentrifuge). </li><li> Überstand entfernen und resuspendieren Zellen mit 2 ml PBS + 2% FCS. </li><li> Filtern Sie die Probe mit einer 70 um Zell-Sieb und sparen Strom durch. </li><li> Zentrifuge bei 4 ° C, 320 xg für 5 min. </li><li> Überstand entfernen und resuspendieren Zellen mit 300 ul PBS + 2% FCS. </li><li> Zählen Sie die Zellen (ca. 5-10 x 10 5 Zellen sind aus einem Oberkiefer erwartet). </li></ol> 3. Extrazelluläre und intrazelluläre AntikörperFärbung für die Durchflusszytometrie-Analyse Es wird empfohlen, in einer Haube arbeiten, mit Lichter aus. Es ist wichtig, um die Zellen in kalter Umgebung konstant zu halten, sowie alle benötigten Materialien. <ol><li> Stain-Zellen gegen extrazelluläre Moleküle durch Zugabe von 0,2-0,5 ug jedes ausgewählte Antikörper pro 1 x 10 6 Zellen in einem Gesamtvolumen von 100 ul. </li><li> Vortex kurz. </li><li> Die Röhrchen für 15 min im Dunkeln bei 4 ° C. </li><li> Waschen der Zellen mit 2 ml PBS + 2% FCS. </li><li> Zentrifuge bei 4 ° C, 320 xg für 5 min. </li><li> Saugen Sie den Überstand und resuspendieren Zellen durch Zugabe tropfenweise, 500 ul kalten BD Cytoperm / Cytofix unter Vortexen. </li><li> Die Röhrchen für 40 min bei 4 ° C im Dunkeln. </li><li> Vortex kurz. </li><li> Waschen Sie die Zellen zweimal mit 1 ml kaltem BD Perm / Wash Buffer und Spin bei 800 xg für 7 min bei 4 ° C. </li><li> Saugen Sie den Überstand bis auf 300 ul. </li><li> Stain Zellen intrazellulär durchZugabe von 1 ug jedes ausgewählte Antikörper pro 1 x 10 6 Zellen in einem Gesamtvolumen von 100 ul. </li><li> Vortex kurz. </li><li> Inkubieren für 30 min im Dunkeln bei 4 ° C. </li><li> Waschen Sie die Zellen zweimal mit 1 ml kaltem BD Perm / Wash Buffer und Spin bei 800 xg für 7 min bei 4 ° C. </li><li> Saugen Sie den Überstand und resuspendieren Zellen durch Zugabe von 300 ul kalten BD Perm / Wash Buffer. </li><li> Vortex kurz. </li><li> Übertragen Sie die Suspension während filtern FACS-Röhrchen. </li><li> Halten Sie die Rohre in einer dunklen und kalten Umgebung bis zur Analyse. </li></ol>

<ol>
	<li>Arizon, 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=Langerhans+cells+down-regulate+inflammation-driven+alveolar+bone+loss.">Langerhans cells down-regulate inflammation-driven alveolar bone loss.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 7043-7048 (2012).</li><li>Listgarten, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+periodontitis.">Pathogenesis of periodontitis.</a> J. Clin. Periodontol. 13, 418-430 (1986).</li><li>Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causation+and+pathogenesis+of+periodontal+disease.">Causation and pathogenesis of periodontal disease.</a> Periodontol. 25, 8-20 (2000).</li><li>Jotwani, 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=Mature+dendritic+cells+infiltrate+the+T+cell-rich+region+of+oral+mucosa+in+chronic+periodontitis:+in+situ,+in+vivo,+and+in+vitro+studies.">Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.</a> J. Immunol. 167, 4693-4700 (2001).</li><li>Graves, D. T., 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=Interleukin-1+and+tumor+necrosis+factor+antagonists+inhibit+the+progression+of+inflammatory+cell+infiltration+toward+alveolar+bone+in+experimental+periodontitis.">Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis.</a> J. Periodontol. 69, 1419-1425 (1998).</li><li>Kabashima, H., Yoneda, M., Nagata, K., Hirofuji, T., Maeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+chemokine+(MCP-1,+MIP-1alpha,+MIP-1beta,+IP-10,+RANTES)-positive+cells+and+chemokine+receptor+(CCR5,+CXCR3)-positive+cells+in+inflamed+human+gingival+tissues.">The presence of chemokine (MCP-1, MIP-1alpha, MIP-1beta, IP-10, RANTES)-positive cells and chemokine receptor (CCR5, CXCR3)-positive cells in inflamed human gingival tissues.</a> Cytokine. 20, 70-77 (2002).</li><li>Moughal, N. A., Adonogianaki, E., Kinane, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Langerhans+cell+dynamics+in+human+gingiva+during+experimentally+induced+inflammation.">Langerhans cell dynamics in human gingiva during experimentally induced inflammation.</a> J. Biol. Buccale. 20, 163-167 (1992).</li><li>Cochran, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+bone+loss+in+periodontal+disease.">Inflammation and bone loss in periodontal disease.</a> J. Periodontol. 79, 1569-1576 (2008).</li><li>Taubman, M. A., Valverde, P., Han, X., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+response:+the+key+to+bone+resorption+in+periodontal+disease.">Immune response: the key to bone resorption in periodontal disease.</a> J. Periodontol. 76, 2033-2041 (2005).</li><li>Graves, D. T., Kang, J., Andriankaja, O., Wada, K., Rossa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+to+study+host-bacteria+interactions+involved+in+periodontitis.">Animal models to study host-bacteria interactions involved in periodontitis.</a> Front Oral Biol. 15, 117-132 (2012).</li></ol>

Autoren haben nichts zu offenbaren.

Oberkiefer Zahnfleischgeweben aus einem einzigen Mausklick erhalten sind ausreichend für die Analyse von Sub-Populationen von T-und B-Lymphozyten, sowie ihre Fähigkeit, extra-und intrazellulären Molekülen auszudrücken, wie wir zuvor beschrieben 1. Dennoch, wenn seltenen Zellpopulationen von Interesse sind (z. B. DCs), wird es in Pool Geweben 2-3 Mäuse empfohlen. Zu beachten ist, wenn bevorzugt, ist es möglich, sowohl den Gaumen schälen und Zahnfleischgeweben und dann der Verbrauchsteuer das Zahnfleisc...

Gingiva ist das weiche Gewebe um den zervikalen Teil der Zähne und deckt den Alveolarfortsatz (Abbildung 1). Das Zahnfleisch ist eine Art von Kau-Mukosa, die weiter in Schleimhaut-Epithel und Bindegewebe (auch als Submukosa oder Lamina propria bekannt) getrennt werden können. Die anatomische Struktur des Zahnfleisches und der angrenzenden Zähne ermöglicht es Bakterien, leben und entwickeln Plaque (Biofilm), die ständig herausfordert die lokale Immunsystem. Als ein Ergebnis entwickelt Entzündungsreaktion in der Gingiva, die unter Umständen gesundheitsschädlich wird - ein Zustand bezeichnet Parodontopathien 2. Grundsätzlich kann Plaque-induzierte parodontalen Erkrankungen in Gingivitis und Parodontitis unterteilt werden. Gingivitis eine reversible Zustand der lokalen Entzündungsreaktion, die das Zahnfleisch beschränkt ist. Periodontitis, auf der anderen Seite, ist eine irreversible zerstörende Verfahren, bei dem die Befestigungsvorrichtung (Alveolarknochen periodontalenLig. Zement und Zahnfleisch) ist 3 zerstört. Das Zahnfleisch wurde vorgeschlagen, sowohl als Effektor und induktive Websites während Parodontalerkrankungen 4 dienen. Menschliche Studien haben vorgeschlagen, dass als Reaktion auf Zahnbelag, Immuneffektorzellen und Moleküle dynamisch infiltriert oder Abreise das Zahnfleisch 5-7. Diese Aktivität wurde gezeigt, dass eine wichtige Rolle bei der parodontalen Zerstörung 8,9 spielen. Während Daten von diesen Studien lieferten wertvolle Informationen über diesen pathologischen Prozess, die Arbeit mit menschlichen Geweben erzeugt besitzen erhebliche ethische, technische und experimentelle Einschränkungen. Die Entwicklung von experimentellen Modellen erlaubt Ursache-Wirkungs-Experimente über den Einsatz transgener Mäuse und in vivo Interventionen 10. Dadurch erhöht unser Wissen über die Mechanismen in Parodontitis beteiligt sich während der letzten zwei Jahrzehnte. Trotzdem aufgrund der Komplexität der periodontalen Krankheiten bestehteine Debatte über die Art der lokalen Immunantwort Erleichterung Gewebezerstörung. Es ist auch ein Mangel in unserem Verständnis von der Funktion des zentralen Immunzellen in das Zahnfleisch während Parodontalerkrankungen. Es ist somit wesentlich, pathologische entzündliche Ereignisse in das Zielgewebe von der Krankheit, der Gingiva zu studieren.

<table border="1">
 <tbody>
 <tr>
 <td>Comments (optional)</td>
 <td>Catalogue number</td>
 <td>Company</td>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td></td>
 <td>CLS-2</td>
 <td>Worthington Biochemical Corp.</td>
 <td>Collagenase Type II</td>
 </tr>
 <tr>
 <td></td>
 <td>DN25-1G</td>
 <td>SIGMA</td>
 <td>DNAse I</td>
 </tr>
 <tr>
 <td></td>
 <td>E6758-100G</td>
 <td>SIGMA</td>
 <td>EDTA</td>
 </tr>
 <tr>
 <td></td>
 <td>D8537</td>
 <td>SIGMA</td>
 <td>Dulbecco's PBS</td>
 </tr>
 <tr>
 <td>Heat Inactivated</td>
 <td>04-121-1</td>
 <td>Biological Industries</td>
 <td>Fetal Bovine Serum </td>
 </tr>
 <tr>
 <td></td>
 <td>FPE-204-500</td>
 <td>Jet Biofil</td>
 <td>Vacuum-Driven Filter</td>
 </tr>
 <tr>
 <td></td>
 <td>352052</td>
 <td>BD Falcon</td>
 <td>5 ml Polystyrene Round-Bottom Tube</td>
 </tr>
 <tr>
 <td></td>
 <td>93070</td>
 <td>SPL Lifesciences</td>
 <td>Cell Strainer 70 &mu;m</td>
 </tr>
 <tr>
 <td></td>
 <td>153066</td>
 <td>NUNC</td>
 <td>Tissue Culture Dish 35&times;10 mm</td>
 </tr>
 <tr>
 <td></td>
 <td>554714</td>
 <td>BD</td>
 <td>BD Cytofix/Cytoperm</td>
 </tr>
 <tr>
 <td>Clone N418</td>
 <td>117305</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD11c antibody</td>
 </tr>
 <tr>
 <td>Clone 104</td>
 <td>109819</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD45.2 antibody</td>
 </tr>
 <tr>
 <td>Clone GK1.5</td>
 <td>100413</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD4 antibody</td>
 </tr>
 <tr>
 <td>Clone 53-6.7</td>
 <td>100733</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD8a antibody</td>
 </tr>
 <tr>
 <td>Clone 17A2</td>
 <td>100214</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD3 antibody</td>
 </tr>
 <tr>
 <td>Clone G8.8</td>
 <td>118219</td>
 <td>Biolegend</td>
 <td>Anti-mouse CD326 (Ep-CAM) antibody</td>
 </tr>
 <tr>
 <td>Clone 929F3.01</td>
 <td>DDX0362D</td>
 <td>Imgenex</td>
 <td>Anti-mouse CD207 (Langerin) antibody</td>
 </tr>
 <tr>
 <td>Clone 39-10-8</td>
 <td>115010</td>
 <td>Biolegend</td>
 <td>Anti-mouse I-Ad (MHC-II) antobody</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td>BD Biosciences</td>
 <td>LSR II Flow Cytometer</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td>Tree Star</td>
 <td>FlowJo Software v 7.6.5</td>
 </tr>
 </tbody>
</table>

isolation, processing and analysis of murine gingival cells

Wir haben eine Technik entwickelt, um genau zu isolieren und zu verarbeiten Maus Zahnfleisch für die Durchflusszytometrie und molekularbiologische Studien entwickelt. Das Zahnfleisch ist eine einzigartige und wichtige Gewebe Immunmechanismen zu studieren, weil es in Immunantwort gegen orale Biofilme, die Parodontalerkrankungen führen könnten beteiligt ist. Darüber hinaus ermöglicht die Nähe des Zahnfleisches zu Alveolarknochen Gewebe auch das Studium Knochenumbau unter entzündlichen Bedingungen. Unsere Methode liefert große Mengen von Immunzellen, die Analyse selbst seltenen Zellpopulationen wie Langerhans-Zellen und regulatorischen T-Zellen ermöglicht, wie wir bereits gezeigt, 1. Verwendung Mäusen zu lokalen Immunreaktionen in Alveolarknochenschwund bei Parodontopathien beteiligt studieren ist aufgrund der Verfügbarkeit von verschiedenen immunologischen und experimentelle Werkzeuge vorteilhaft. Allerdings lassen sich aufgrund ihrer geringen Größe und der relativ unbequem Zugang zum murinen Zahnfleisch, vermieden viele Studien Prüfung von this kritischen Gewebe. Das Verfahren in dieser Arbeit beschriebenen erleichtern könnte gingivalen Analyse, die hoffentlich unsere Unterbewertung auf die orale Immunsystem und seine Rolle während der Parodontalerkrankungen wird zunehmen.

Beispiele für Durchflusszytometrie-Analyse auf Gingivazellen vorgestellt. Gingival Zellen aus 2 Mäuse wurden vereinigt in einem LSR II Durchflusszytometer laufen und unter Verwendung FlowJo Software. 3A demonstriert die Verteilung der gingivalen Zellen aus naiven Mäusen in einer Seitwärts-Streulicht (SSC) gegen forward scatter (FSC) Grundstück. Gating Strategie zur Identifizierung (i) Lymphozyten (ii) Monozyten / dendritische Zellen und (iii) Granulozyten angegeben. Zum Vergleich dazu präsentieren...

Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

Isolation, Processing and Analysis of Murine Gingival Cells

Diese Studie beschreibt ein effizientes Verfahren zur Isolation und Behandlung Gingiva von der Maus Mundhöhle, um eine Einzelzellen-Kultur zu erzeugen. Die resultierenden Zellen können weiter für die Durchflusszytometrie und molekulare Studien eingesetzt werden.

Isolation, Verarbeitung und Analyse von Murine Gingivazellen

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

isolation-processing-and-analysis-of-murine-gingival-cells

Research

JoVE Journal

Immunology and Infection

18.7K Aufrufe.  Hebrew University - Hadassah Medical Center. Diese Studie beschreibt ein effizientes Verfahren zur Isolation und Behandlung Gingiva von der Maus Mundhöhle, um eine Einzelzellen-Kultur zu erzeugen. Die resultierenden Zellen können weiter für die Durchflusszytometrie und molekulare Studien eingesetzt werden.

Serie: Isolation, Processing and Analysis of Murine Gingival Cells

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Durchflusszytometrie-Analyse von Immunzellen innerhalb Murine Aorten

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Forschung

Immunologie und Infektion

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolierung, Identifizierung und Reinigung von Murine Thymusepithelzellen

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolierung von murinen Lymphknoten Stromazellen

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolierung von Murine Peritonealmakrophagen zur Durchführung Genexpressionsanalyse Nach Toll-like Rezeptoren Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolierung und Analyse über Durchflusszytometrie von Gliom-Infiltration peripheren mononukleären Blutzellen

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolierung, Charakterisierung und funktionelle Untersuchung des Zahnfleisch Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolation und Aktivierung von murinen Lymphozyten

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolierung und Analyse über Durchflusszytometrie der Menschen Endozervikale Gamma-Delta-T-Zellen

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Verdauung der murinen Leber für eine durchflusszytometrischen Analyse der lymphatische Endothelial Zellen

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Analyse der Scherung strömungsinduzierte Migration der Murine Marginal Zone B-Zellen In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...