Authors were funded in part by the intramural program of NIDCR (N.M.M) and supported by a Wellcome Trust Stepping Stones Fellowship (097820/Z/11/B to J.E.K) and by a Manchester Collaborative Centre for Inflammation Research grant (to J.E.K). The authors thank Teresa Wild for critically reviewing the manuscript.

Alle experimentellen Verfahren in diesem Protokoll beschrieben gefolgt erforderlichen Richtlinien und wurden von der Institutional Animal Care und Use Committee, NIDCR / NIH genehmigt. 1. Bereiten Sie im Voraus <ol><li> Bereiten Sie die komplette Medien: RPMI mit 2 mM L-Glutamin, 100 Einheiten / ml Penicillin, 100 ug / ml Streptomycin und 10% FBS. </li><li> Bereiten DNase Mittel: 50 ml RPMI-Medium mit 7,5 ug DNase ergänzt (Make frisch, auf Eis halten zu allen Zeiten). </li><li> Bereiten Collagenase-DNase Medien: 5 ml DNase Medium plus 3,2 mg / ml Collagenase Typ IV (Make frisch, auf Eis halten zu allen Zeiten). </li><li> Pre-kühlen FACS-Puffer: 0,5% FBS in PBS verdünnt. </li><li> Pre-kühlen Zentrifuge bis 4 ° C. </li><li> Warm Schüttelinkubator bis 37 ° C. </li></ol> 2. Isolierung, Dissection und Zellisolierung von Zahnfleischblöcke <ol><li> Euthanize Mäuse von ihnen auszusetzen 2 in einer entsprechenden Kammer zu CO, nach ARAC Richtlinien. </li> &lt;li&gt; Öffnen Sie die Brust- und Bauchhöhle, das Herz und die inneren Organe freizulegen. Zu perfundieren, stellen einen Einschnitt in die untere Hohlvene und perfundieren 3 ml PBS über dem linken Ventrikel unter Verwendung einer 3 ml-Spritze mit einer 27 G Nadel. </li><li> Immobilisieren den Körper und Kopf der Maus auf einen Block mit Magen nach oben zeigt. </li><li> Schneiden Sie jede Kommissur der Lippe in Richtung des Halses mit einer Schere, trennt die Haut, die Unterkiefer und Hals-Region umfasst, Freilegen der darunter liegenden Gewebe und Muskeln. Präparieren Sie die Unterlippe des Unterkiefers zu entdecken. </li><li> Schneiden zwischen den unteren Schneidezähnen auf beiden Seiten des Unterkiefers zu trennen und jede Seite immobilisieren, um die Mundhöhle gelangen. </li><li> Visualisieren und sezieren den Gaumen von der Nasenhöhle. </li><li> Sezieren Bereiche mit einem 10 Blatt und sind Ober- und Unterkieferbackenzahn Bereiche mit umgebenden Zahnfleischentzündung (Abbildung 1). Stellen vertikale Einschnitte gerade anterior zu den ersten Molaren und hinter der dritten Molaren und einer horizontalen Schnitt andie Grenze des Zahnfleisches (white collar des umgebenden Gewebes Zähne). </li><li> Legen Sie die Ober- und Unterkieferblöcke in einem 50 ml konischen Röhrchen mit 5 ml Collagenase / DNase (auf Eis halten). </li><li> Inkubieren in einem Schüttelinkubator bei 37 ° C für 1 Stunde und während der letzten 5 Minuten Inkubation werden 50 ul 0,5 M EDTA. </li><li> In 5 ml kaltem DNase Medien auf die 50-ml-Tube mit Geweben und sanft zu mischen wirbeln. </li><li> Übertragen Sie die vier Blöcke von Gewebe auf eine Petrischale geben und mit 500 ul DNase Medien. Entfernen Zahnfleisch aus jedem Block von Gewebe, eine Skalpellklinge mit und zeigt die Klinge in den Zahnfleisch und Interdentalräume. </li><li> Übertragen die Gewebe und die Medien aus der Petrischale sowie den vorhergehenden DNase Medien bei der Präparation verwendet werden, auf eine neue 50-ml-Röhrchen durch die Zelle Sieb (70 &amp; mgr; m groß). Wasche mit DNase Medien (3-5 ml) alle Instrumente verwendet und filtriert. </li><li> Mit dem Kolben einer sterilen 3 ml Spritze, Maische, das Gewebe gegen das Sieb,Filterung mit kaltem DNase Medien (30 bis 35 ml). </li><li> Waschen Sie die Zelle Sieb mit kaltem DNase Medien. </li><li> Zentrifuge bei 4 ° C, 314 xg für 6 min. </li><li> Überstand verwerfen, erneut zu suspendieren in 1 ml Vollmedien. </li><li> Zählen von Zellen unter Verwendung eines automatischen Zellzählers (die erwartete Menge sollte zwischen 8 x 10 5 bis 1,2 x 10 6 Zellen mit einer Lebensfähigkeit von&gt; 80% liegen). </li></ol> 3. Die Stimulation und FACS-Färbung von Zahnfleisch Cells <ol><li> Stimulieren die Einzelzellsuspension mit PMA (50 ng / ml) und Ionomycin (2,5 &amp; mgr; g / ml) in Gegenwart von 1 ul / ml Brefeldin A. </li><li> Inkubieren für 3,5 Stunden bei 37 ° C und 5% CO 2. </li><li> Spin down und waschen mit PBS und Zentrifuge bei 4 ° C; 314 g für 6 min. </li><li> Stain die Zellen eine Lebensfähigkeit Fleck unter Verwendung nach den Anweisungen des Herstellers. </li><li> Stain für extrazelluläre Marker (CD45, TCRγδ, TCR &amp; bgr;, CD4, CD8, TCRγδ oder Ameiseibodies der Wahl) nach den Anweisungen des Herstellers. </li><li> Waschen Sie die Zellen mit kaltem FACS-Puffer und Zentrifuge bei 4 ° C, 314 × g für 5 min. </li><li> Sanft vortexen das Zellpellet zu stören. </li><li> Fix die Zellen mit 2% Paraformaldehyd für 20 min bei Raumtemperatur. </li><li> Waschen Sie die Zellen mit kaltem FACS-Puffer, Zentrifuge bei 4 ° C, 314 xg für 5 min und Flecken zur intrazellulären Zytokine. <ol><li> Füllt die FACS-Röhrchen mit einer Lösung aus kaltem 0,5% Saponin verdünnt in FACS-Puffer und Zentrifuge bei 4 ° C, 314 × g für 5 min. </li><li> Hinzufügen von 300 &amp; mgr; l von 0,5% Saponin Lösung für die FACS-Röhrchen, Inkubieren für 15 min im Dunkeln bei 4 ° C und dann zentrifugiert bei 4 ° C, 314 × g für 5 min. </li><li> Stain die Zellen für die intrazelluläre Marker (IL-17A und IFNy oder Cytokine der Wahl) nach den Anweisungen des Herstellers. Waschen Sie die Zellen mit 0,5% Saponin-Lösung und Zentrifuge bei 4 ° C, 314 × g für 5 min. </li><li> Waschen Sie wieder mit FACSPuffer und Zentrifuge bei 4 ° C, 314 × g für 5 min. </li><li> Wieder die Zellen in 300 ul FACS-Puffer. </li></ol></li><li> Analysieren Sie die Ergebnisse auf einem Durchflusszytometer. </li></ol> 4. Durchflusszytometrie Analyse <ol><li> Visualisieren Zellen analysiert auf zukunfts (FSC) und Seitenstreuung (SCC) und Gate werden Trümmer auszuschließen. </li><li> Wählen Sie einzelne Zellen mit Vorwärtsstreubereich (FSC-A) vs. Höhe (FSC-H) Parameter. </li><li> Gate-Zellen, die unter Spannung stehen (wie von Lebensfähigkeit Färbung angedeutet) und positiv für die hämatopoetischen Marker CD45 + für weitere Analysen (2A und 2C). </li><li> Weiter Analyse von spezifischen Markern wie in 2 und 3 dargestellt. </li></ol>

<ol>
	<li>Aas, J. A., Paster, B. J., Stokes, L. N., Olsen, I., Dewhirst, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+The+Normal+Bacterial+Flora+Of+The+Oral+Cavity.">Defining The Normal Bacterial Flora Of The Oral Cavity.</a> J Clin Microbiol. 43, 5721-5732 (2005).</li><li>Belkaid, Y., Naik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+And+Systemic+Control+Of+Tissue+Immunity+By+Commensals.">Compartmentalized And Systemic Control Of Tissue Immunity By Commensals.</a> Nat Immunol. 14, 646-653 (2013).</li><li>Darveau, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontitis:+A+Polymicrobial+Disruption+Of+Host+Homeostasis.">Periodontitis: A Polymicrobial Disruption Of Host Homeostasis.</a> Nat Rev Microbiol. 8, 481-490 (2010).</li><li>Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomicrobial+Pathogenesis+Of+Periodontitis:+Keystones,+Pathobionts,+And+Host+Response.">Immunomicrobial Pathogenesis Of Periodontitis: Keystones, Pathobionts, And Host Response.</a> Trends Immunol. 35, 3-11 (2014).</li><li>Eke, P. 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=Prevalence+Of+Periodontitis+In+Adults+In+The+United+States:+2009+And+2010.">Prevalence Of Periodontitis In Adults In The United States: 2009 And 2010.</a> J Dent Res. 91, 914-920 (2012).</li><li>Moutsopoulos, N. M., Lionakis, M. S., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inborn+Errors+In+Immunity:+Unique+Natural+Models+To+Dissect+Oral+Immunity.">Inborn Errors In Immunity: Unique Natural Models To Dissect Oral Immunity.</a> J Dent Res. , (2015).</li><li>Hajishengallis, G., Lamont, R. J., Graves, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Enduring+Importance+Of+Animal+Modelsin+Understanding+Periodontal+Disease.">The Enduring Importance Of Animal Modelsin Understanding Periodontal Disease.</a> Virulence. 6, 229-235 (2015).</li><li>Sharrow, S. O., Coligan, J. E., 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=Analysis+Of+Flow+Cytometry+Data.">Analysis Of Flow Cytometry Data.</a> Current Protocols In Immunology. , (2001).</li><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>Moutsopoulos, N. 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=Defective+Neutrophil+Recruitment+In+Leukocyte+Adhesion+Deficiency+Type+I+Disease+Causes+Local+IL-17-Driven+Inflammatory+Bone+Loss.">Defective Neutrophil Recruitment In Leukocyte Adhesion Deficiency Type I Disease Causes Local IL-17-Driven Inflammatory Bone Loss.</a> Sci Transl Med. 6, 229-240 (2014).</li><li>Gaffen, S. L., Jain, R., Garg, A. V., Cua, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL-23-IL-17+Immune+Axis:+From+Mechanisms+To+Therapeutic+Testing.">The IL-23-IL-17 Immune Axis: From Mechanisms To Therapeutic Testing.</a> Nat Rev Immunol. 14, 585-600 (2014).</li></ol>

Authors have nothing to disclose

Die aktuelle Technik erfolgreich eine große Zahl von Immunzellen führt (aus einer einzigen Maus) geeignet, nicht nur für die phänotypische Charakterisierung, sondern auch für funktionelle Studien ex vivo. Eine andere Gruppe war zuvor ein Protokoll veröffentlicht zu isolieren und murine gingival Immunzellen zu charakterisieren und stellte den Wert der mehrfarbigen mit Durchflusszytometrie in der Studie von Parodontitis in Tiermodellen 9. Nach erheblichen Fehlersuche den Schlüssel Änderung in d...

Zahnfleischgewebe umgeben den Menschen und der Maus Gebiss und werden ständig auf die komplexe Biofilm des Zahnes 1 ausgesetzt. Die Immunzellennetz der Zahnfleischschutz Überwachung ist entscheidend für Gewebeintegrität zu gewährleisten, mit den lokalen kommensale Bakterien gewährleisten Homöostase und, zur gleichen Zeit, zwei wirksame Immunität gegen pathogene Herausforderung bietet. Um das zu erreichen Homöostase wird das Immunsystem sorgfältig auf die Zahnfleischumgebung zugeschnitten eine hoch spezialisierte Immunzellen Netzwerk zu schaffen, noch so kleine Detail wird von den Zahnfleischimmunzellpopulationen bekannt und ihre Rolle bei der Aufrechterhaltung der Gewebe Immunität 2. Wenn Immunhomöostase auf dem Zahnfleisch gestört ist, entweder durch eine erhöhte Anfälligkeit Host und / oder das Vorhandensein von dysbiotischen mikrobiellen Gemeinschaften, eine entzündliche Erkrankung entsteht Parodontitis 3 4. Parodontitis ist eine weitverbreitete entzündliche Erkrankung, was zu einem Verlust des Zahnes supporting Strukturen. In seiner schweren Formen wird in ca. 10% der Bevölkerung im allgemeinen 5 ersichtlich. Die Schlüsselfaktoren in Parodontitis Anfälligkeit und Progression beteiligt Sezieren hat sich als schwierig erwiesen, 6. Allerdings Tiermodellen haben im Verständnis der Mechanismen der Parodontitis Initiierung und Progression 7 äußerst nützlich. Die Modelle können die wichtigsten Zellpopulationen und molekulare Mechanismen zu definieren, verwendet werden, die Immunhomöostase und Antrieb sind entscheidend Entwicklung einer Parodontitis zu halten. Eine solche Erkenntnis wird unser Verständnis von Zahnfleisch-spezifische Steuerung von Immunhomöostase und weiterhin unser gegenwärtiges Verständnis der Krankheitsentstehung zu transformieren.

<table border="0" cellpadding="0" cellspacing="0" width="914">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fine Scissors</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">14058-11</td>
			<td style="width:235px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Scalpel Handle #3</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">10003-12</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Scalpel Blades #10</td>
			<td style="width:188px;">Fine science tools</td>
			<td style="width:188px;">10010-00</td>
			<td>Sterile</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Splinter Forceps</td>
			<td style="width:188px;">Integra Miltex</td>
			<td style="width:188px;">6-304</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Needles with regular bevel&#160;</td>
			<td style="width:188px;">BD Medical</td>
			<td style="width:188px;">305109</td>
			<td>27G, 12.7 mm length</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Monoject syringes</td>
			<td style="width:188px;">Covidien</td>
			<td style="width:188px;">8881513934</td>
			<td>Luer-lock tip, 3mL</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">PBS, pH 7.4</td>
			<td style="width:188px;">Life Technologies</td>
			<td style="width:188px;">10010-049</td>
			<td>Without Calcium and Magnesium</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">RPMI 1640</td>
			<td style="width:188px;">Lonza</td>
			<td style="width:188px;">12-167F</td>
			<td>Without L-glutamine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">DNase I from bovine pancreas</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">DN25-1G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:304px;">Collagenase type IV</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">17104-019</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Fetal Bovine Serum</td>
			<td style="width:188px;">Gemini Bio-products</td>
			<td style="width:188px;">100-106</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Gentamicin 50 mg/ml</td>
			<td style="width:188px;">Quality biological</td>
			<td style="width:188px;">120-098-661EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:304px;">Pen Strep</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">15140-122</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:304px;">L-Glutamine</td>
			<td style="width:188px;">Gibco (by Life technologies)</td>
			<td style="width:188px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">0.5M EDTA pH 8.0</td>
			<td style="width:188px;">Quality biological</td>
			<td style="width:188px;">351-027-721EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">50 mL tubes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352070</td>
			<td>Polypropylene, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">70 &#956;M Cell Strainers</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352350</td>
			<td></td>
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			<td height="21" style="height:21px;width:304px;">Petri dishes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">351029</td>
			<td>Sterile</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">5 mL FACS tubes</td>
			<td style="width:188px;">Corning</td>
			<td style="width:188px;">352052</td>
			<td>Sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD GolgiPlug</td>
			<td style="width:188px;">BD Biosciences</td>
			<td style="width:188px;">555029</td>
			<td>Contains brefeldin A solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Phorbol 12-Myristate 13-Acetate (PMA)</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">P8139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Ionomycin Calcium Salt</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">13909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Saponin from quillaja bark</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:188px;">S4521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Aqua Dead Cell Stain Kit</td>
			<td style="width:188px;">Life Technologies</td>
			<td>L34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD45 Alexa Fluor 700</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">56-0451-82</td>
			<td></td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD4 eFluor 450</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">48-0042-82</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse TCR beta APC eFluor 780</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">47-5961-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse gamma delta TCR FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5711-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse IL-17A APC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">12-7311-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse IFN-&#947; PE</td>
			<td style="width:188px;">eBioscience</td>
			<td>11-5931-82</td>
			<td></td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse NK1.1 PE-Cy7</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">25-5941-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD90.2 APC eFluor 780</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">47-0902-82</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD3e FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0031-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD19 FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0193-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11b FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0112-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse CD11c FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-0114-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse TCR beta FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5961-82</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:304px;">Anti-Mouse Ly-6G FITC</td>
			<td style="width:188px;">eBioscience</td>
			<td style="width:188px;">11-5931-82</td>
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			<td height="21" style="height:21px;width:304px;">Anti-Mouse Ly-6C FITC</td>
			<td style="width:188px;">BD Pharmingen</td>
			<td style="width:188px;">553104</td>
			<td></td>
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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.

Zur Anwendung des Protokolls veranschaulichen, zeigen wir repräsentative Ergebnisse der Immunzellennetz in das Zahnfleisch von Mäusen mit und ohne Parodontitis untersucht (WT vs. LFA - / - 2A - C). Repräsentative FACS Plots zeigen Live-CD45 + hämatopoetische Zellen im Zahnfleisch (2A, 2C). Isolation und Verarbeitung von Immunzellen mit diesem Protokoll liefert genügend Zellen ex vivo S...

Watch this Scientific Journal Video about Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network at JoVE.com

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

Wir haben ein Verfahren zur Isolierung, phänotypische Charakterisierung und funktionelle Analyse von Immunzellen aus murinen Gingiva etabliert.

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-characterization-functional-examination-gingival-immune

Research

JoVE Journal

Immunology and Infection

10.7K Aufrufe.  NIH.  Wir haben ein Verfahren zur Isolierung, phänotypische Charakterisierung und funktionelle Analyse von Immunzellen aus murinen Gingiva etabliert. 

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

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Forschung

Immunologie und Infektion

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolierung von Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Differenziert Functional Roles of Gene Expression von Immun-und Nicht-Immunzellen in Mouse Colitis von Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Durchflusszytometrische Isolation of Primary Murine Type II Alveolarepithelzellen für Funktionelle und Molekulare Studien

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

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 of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Isolation von Fettgewebe Immune Cells

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Ein<em&gt; In-vitro-</em&gt; Modell zur Messung der Immunantwort auf Malaria im Kontext der HIV-Koinfektion

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolation und Analyse über Durchflusszytometrie von Immunzellen aus dem ischämischen Gehirn der Maus

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Isolierung und Charakterisierung von Neutrophilen abgeleitet Mikropartikel für funktionelle Studien

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.