Name: isolation, processing and analysis of murine gingival cells
Uploaded: 2013-07-02T14:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Isolation, Processing and Analysis of Murine Gingival Cells at JoVE.com

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><tbody><tr><td>Collagenase Type II</td><td>Worthington Biochemical Corp.</td><td>CLS-2</td><td></td></tr><tr><td>DNAse I</td><td>SIGMA</td><td>DN25-1G</td><td></td></tr><tr><td>EDTA</td><td>SIGMA</td><td>E6758-100G</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>SIGMA</td><td>D8537</td><td></td></tr><tr><td>Fetal Bovine Serum </td><td>Biological Industries</td><td>04-121-1</td><td>Heat Inactivated</td></tr><tr><td>Vacuum-Driven Filter</td><td>Jet Biofil</td><td>FPE-204-500</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube</td><td>BD Falcon</td><td>352052</td><td></td></tr><tr><td>Cell Strainer 70 &amp;mu;m</td><td>SPL Lifesciences</td><td>93070</td><td></td></tr><tr><td>Tissue Culture Dish 35&amp;#215;10 mm</td><td>NUNC</td><td>153066</td><td></td></tr><tr><td>BD Cytofix/Cytoperm</td><td>BD</td><td>554714</td><td></td></tr><tr><td>Anti-mouse CD11c antibody</td><td>Biolegend</td><td>117305</td><td>Clone N418</td></tr><tr><td>Anti-mouse CD45.2 antibody</td><td>Biolegend</td><td>109819</td><td>Clone 104</td></tr><tr><td>Anti-mouse CD4 antibody</td><td>Biolegend</td><td>100413</td><td>Clone GK1.5</td></tr><tr><td>Anti-mouse CD8a antibody</td><td>Biolegend</td><td>100733</td><td>Clone 53-6.7</td></tr><tr><td>Anti-mouse CD3 antibody</td><td>Biolegend</td><td>100214</td><td>Clone 17A2</td></tr><tr><td>Anti-mouse CD326 (Ep-CAM) antibody</td><td>Biolegend</td><td>118219</td><td>Clone G8.8</td></tr><tr><td>Anti-mouse CD207 (Langerin) antibody</td><td>Imgenex</td><td>DDX0362D</td><td>Clone 929F3.01</td></tr><tr><td>Anti-mouse I-A&lt;sup&gt;d&lt;/sup&gt; (MHC-II) antobody</td><td>Biolegend</td><td>115010</td><td>Clone 39-10-8</td></tr><tr><td>LSR II Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo Software v 7.6.5</td><td>Tree Star</td><td></td><td></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

18.8K 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

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 ...

Forschung

Immunologie und Infektion

Rapid Isolation of Single Cells from Mouse and Human Teeth

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental pulp tissue, rich in the extracellular matrix, requires a long and tedious dissociation process that is typically beyond the reasonable time for single-cell transcriptomics. For avoiding artificial changes in gene expression, the time elapsed from euthanizing an animal until the analysis of single cells needs to be minimized. This work presents a fast protocol enabling to obtain single-cell suspension from mouse and human teeth in an excellent quality suitable for scRNA-seq (single-cell RNA-sequencing). This protocol is based on accelerated tissue isolation steps, enzymatic digestion, and subsequent preparation of final single-cell suspension. This enables fast and gentle processing of tissues and allows using more animal or human samples for obtaining cell suspensions with high viability and minimal transcriptional changes. It is anticipated that this protocol might guide researchers interested in performing the scRNA-seq not only on the mouse or human teeth but also on other extracellular matrix-rich tissues, including cartilage, dense connective tissue, and dermis.

The current protocol presents a fast, efficient, and gentle method for isolating single cells suitable for single-cell RNA-seq analysis from a continuously growing mouse incisor, mouse molar, and human teeth.

Schnelle Isolierung einzelner Zellen aus Maus- und menschlichen Zähnen

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...

Entwicklungsbiologie

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Isolierung von Maus Speicheldrüsen Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Biologie

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

Robuste Ligatur-induzierte Modell der Murine Parodontitis für die Bewertung von oralen Neutrophilen

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

Isolierung und Kultur primärer humaner Zahnfleischepithelzellen mit Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Biotechnik

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissektion und Isolierung von murinen Glia aus mehreren Regionen des zentralen Nervensystems

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

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Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Die Isolierung von murinen Coronary glatten Gefäßmuskelzellen

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

Isolierung von myepithelialen Zellen aus der Urmorine, Lacrimal und Submandibular Drüsen

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
This video demonstrates a technique for assessing the ability of Porphyromonas gingivalis, an anaerobic pathogenic bacterium, to adhere to and invade human umbilical vein endothelial cells (HUVECs). After infecting the host cells with the bacteria, the host cells are lysed to release both adhered and internalized bacteria. When the lysate is plated in an appropriate medium, the presence of colonies indicates successful bacteria-host cell interactions.

Ein bakterieller Infektionsassay zur Untersuchung anaerober Bakterien-Wirtszell-Interaktionen

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols ...