Wir sind dankbar, dass die Chirurgen der Klinik für Thorax-und Kardiovaskuläre Chirurgie, Universitätsklinik Tübingen für die Bereitstellung der Proben Thymus und Bruno Kyewski (DKFZ, Heidelberg, Deutschland) für die Bereitstellung der CDR2-Antikörper. Wir möchten auch an Hans-Jörg Bühring und Sabrina Grimm von der Sortieranlage (Universität Tübingen) danken. Diese Arbeit wurde durch den SFB 685 und der Hertie-Stiftung unterstützt.

<ol>
	<li>Miller, J. F. A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+of+thymus+function+and+of+thymus-derived+lymphocytes.">The discovery of thymus function and of thymus-derived lymphocytes.</a> Immunological Reviews. 185, 7-14 (2002).</li><li>Klein, L., Hinterberger, M., Wirnsberger, G., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+in+the+thymus+for+positive+selection+and+central+tolerance+induction.">Antigen presentation in the thymus for positive selection and central tolerance induction.</a> Nat Rev Immunol. 9, 833-844 (2009).</li><li>Vandenabeele, S., Hochrein, H., Mavaddat, N., Winkel, K., Shortman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+thymus+contains+2+distinct+dendritic+cell+populations.">Human thymus contains 2 distinct dendritic cell populations.</a> Blood. 97, 1733-1741 (2001).</li><li>Gotter, J., Brors, B., Hergenhahn, M., Kyewski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medullary+Epithelial+Cells+of+the+Human+Thymus+Express+a+Highly+Diverse+Selection+of+Tissue-specific+Genes+Colocalized+in+Chromosomal+Clusters.">Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters.</a> The Journal of Experimental Medicine. 199, 155-166 (2004).</li><li>Rouse, R. V., Bolin, L. M., Bender, J. R., Kyewski, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+reactive+with+subsets+of+mouse+and+human+thymic+epithelial+cells.">Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells.</a> Journal of Histochemistry &amp; Cytochemistry. 36, 1511-1517 (1988).</li><li>Stoeckle, C., 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=Cathepsin+S+dominates+autoantigen+processing+in+human+thymic+dendritic+cells.">Cathepsin S dominates autoantigen processing in human thymic dendritic cells.</a> Journal of Autoimmunity. 38, 332-343 (2012).</li><li>Woods Ignatoski, K. M., Bingham, E. L., Frome, L. K., Doherty, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+trans-differentiation+of+thymus+cells+into+parathyroid-like+cells+without+genetic+manipulation.">Directed trans-differentiation of thymus cells into parathyroid-like cells without genetic manipulation.</a> Tissue Eng Part C Methods. 17, 1051-1059 (2011).</li><li>Williams, K. 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=Single+Cell+Analysis+of+Complex+Thymus+Stromal+Cell+Populations:+Rapid+Thymic+Epithelia+Preparation+Characterizes+Radiation+Injury.">Single Cell Analysis of Complex Thymus Stromal Cell Populations: Rapid Thymic Epithelia Preparation Characterizes Radiation Injury.</a> Clinical and Translational Science. 2, 279-285 (2009).</li><li>Bendriss-Vermare, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+thymus+contains+IFN-&#945;-producing+CD11c-,+myeloid+CD11c+,+and+mature+interdigitating+dendritic+cells.">Human thymus contains IFN-&#945;-producing CD11c-, myeloid CD11c+, and mature interdigitating dendritic cells.</a> The Journal of Clinical Investigation. 107, 835-844 (2001).</li><li>Schmitt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+characterization+of+human+thymic+dendritic+cell+subsets.">Ex vivo characterization of human thymic dendritic cell subsets.</a> Immunobiology. 212, 167-177 (2007).</li><li>Dzionek, A., 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=BDCA-4:+Three+Markers+for+Distinct+Subsets+of+Dendritic+Cells+in+Human+Peripheral+Blood.">BDCA-4: Three Markers for Distinct Subsets of Dendritic Cells in Human Peripheral Blood.</a> The Journal of Immunology. 165, 6037-6046 (2000).</li><li>Wu, L., Shortman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+thymic+dendritic+cells.">Heterogeneity of thymic dendritic cells.</a> Seminars in Immunology. 17, 304-312 (2005).</li><li>Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purified+enzymes+improve+isolation+and+characterization+of+the+adult+thymic+epithelium.">Purified enzymes improve isolation and characterization of the adult thymic epithelium.</a> Journal of Immunological Methods. 385, 23-34 (2012).</li><li>Adamopoulou, E., Tenzer, S., Hillen, N., Klug, P., Rota, I. A., Tietz, S., Gebhardt, M., Stevanovic, S., Schild, H., 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=Exploring+the+MHC-peptide+matrix+of+central+tolerance+in+the+human+thymus.">Exploring the MHC-peptide matrix of central tolerance in the human thymus.</a> Nature Communications. , (2013).</li></ol>

Kein Interessenkonflikt erklärt.

Das hier beschriebene Protokoll ist eine Modifikation des Protokolls durch Gotters et al 4 veröffentlicht. Kritischen Schritte in dem Protokoll sind der Zustand und die ursprüngliche Herstellung des Gewebes sowie der Percoll-Dichte-Trennung. Wir empfehlen dringend, das Gewebe so bald wie möglich nach der Entnahme zu verarbeiten. Es ist wichtig, schnell, aber gründlich zu arbeiten, wenn die Reinigung und das Schneiden des Gewebes. Während der in Schritt 2.3 beschrieben thymocyte waschen, ist es e...

Der Thymus ist das Organ, in dem T-Zell-Entwicklung auftritt. Seine relative und absolute Größe mit dem Alter abnimmt, wenn sie teil sukzessive durch Fett ersetzt, obwohl Thymusaktivität noch im Alter festgestellt werden. Seine Bedeutung für die Immunantwort wurde in den frühen 1960er Jahren ein demonstriert. Die T-Zell-Repertoire ist durch die Wechselwirkung der T-Zell-Rezeptoren, die mit Peptid-MHC-Komplexe auf unterschiedliche Arten von Thymus-APC, die das Überleben oder Tod Signale zu entwickelnden T-Zellen, was zu einer funktionellen und weitgehend selbst tolerant T-Zell-Repertoire 2 vorzusehen geformt. Etwa 98% der Zellen im menschlichen Thymus-T-Zellen entwickeln sich als Thymozyten bezeichnet. Die restlichen 2% bestehen aus einer Reihe von verschiedenen Zelltypen, einschließlich einer Vielzahl von TEC (kortikalen Mark, subkapsuläre), myeloide und plasmazytoiden DC (MDC, PDC), Makrophagen, B-Zellen reifen Umlauf-T-Zellen, Granulozyten, fibroblasts, Endothelzellen und Epithelzellen seltenen Zellen mit einem Expressions Phänotyp ähnlich derjenigen Zellen aus anderen Geweben, wie Muskel, Nervenzellen und respiratorischen Epithels (Abbildung 1). Davon TEC und DC sind die wichtigsten Arten von APC in einem normalen Thymus gefunden. In den letzten Jahren ist die Reinigung dieser APC-Typen für Kultur und molekularen Charakterisierung mehr und mehr an Bedeutung gewonnen. Aufgrund ihrer niedrigen Frequenz, Isolierung jeder dieser Zelltypen für eine detaillierte Analyse erfordert eine effiziente, reproduzierbare und kostengünstiges Verfahren. Die hier vorgestellte Methode ist eine Modifikation von bisher veröffentlichten Studien 3,4. Wie bei jedem anderen Gewebe können Zellgewinnung aus dem Thymus von enzymatisch Disaggregation der Zell-Zell-und Zell-Matrix-Interaktionsnetzwerke, um eine Suspension von Einzelzellen zu erhalten, erreicht werden. Es gibt bestimmte Parameter, wie gut Dissoziation Effizienz, Zellausbeute, die Lebensfähigkeit der Zellen und die Aufrechterhaltung des Zell surface Marker, die von entscheidender Bedeutung sind, und müssen für die erfolgreiche Isolierung dieser seltenen Zellpopulationen optimiert werden. In diesem Protokoll wird Trennung von DC und TEC Teilmengen, indem eine Einzelzell-Suspension aus dem Gewebe durch mechanische Zerstörung und enzymatischen Verdau durchgeführt. Wir verwenden Collagenase aus Clostridium histolyticum A, die ein ausgewogenes Verhältnis von verschiedenen Enzymaktivitäten hat, um die native Kollagen, die das Gewebe zusammenhält brechen. DNase I in der Enzymlösung enthalten, um die Zellaggregation durch freie DNA von toten Zellen zu reduzieren (Thymozyten sind sehr empfindlich). Wir bieten auch einen alternativen Ansatz zu den typischen enzymatische Verdauung Gewebe mit mechanischen und enzymatischen Gewebebehandlung durch eine Gewebe Dissoziator unterstützt. Die Einzelzellsuspension wird dann in einer einzigen Percoll-Dichte-Zentrifugation zur Anreicherung der Fraktion mit niedriger Dichte (LDF) von Zellen unterzogen. Von diesem Teil der Zellen kann durch Färbung DC f isoliert werdenoder DC-Oberflächenmarker (dh CD11c +) und unter Verwendung magnetische Trennung oder Fluoreszenz-aktivierte Zellsortierung (FACS). Im Gegensatz zu den lymphoiden Zellen, die große Mehrheit der Zellen in der Thymusdrüse, nicht TEC nicht exprimieren CD45 auf hohem Niveau, jedoch sind positiv für das epitheliale Zelladhäsionsmolekül EpCAM. cTEC aus Mark TEC durch den Ausdruck eines noch nicht definierten Antigen durch die CDR-2 (kortikale dendritischen Retikulozyten-2)-Antikörper 4,5 und etwas niedriger EpCAM-Expression erkannt unterschieden werden. Der Differenz Co-Expression von EpCAM und CDR2 ermöglicht die effiziente Isolierung dieser TEC Teilmengen über Hochgeschwindigkeits-Zellsortierung 6. Die hier vorgestellte Protokoll ist für den menschlichen Thymus-Gewebe optimiert. Die Dauer des Verfahrens hängt von der Menge des Gewebes und der Fähigkeit der Versuchs sowie der Geschwindigkeit des Zellsortierer, wenn FACS-Sortierung verwendet. Normalerweise kann das Protokoll für die Isolierung von DC in 5 abgeschlossen-6 H und für die Isolierung von TEC in 8-10 Std. Die Trennung von DC-und TEC Teilmengen aus Thymus-Gewebe ist an der Zeit empfindlich. Je schneller die Isolierungsverfahren, die besser der Zustand der Zellen. Schließlich können die isolierten Zellen für weitere Untersuchungen, wie Vergleichsstudien von mRNA und Proteinexpression, PCR-Experimente, Proteinisolierung, molekularen Charakterisierung (dh Transkriptom, Mikro-RNA-Analyse) als auch Zellkultur 6 verwendet werden. Ethikerklärung Um in der Lage, mit der menschlichen Thymusgewebe arbeiten muss der Forscher der Zustimmung der lokalen Ethikkommission oder zuständigen Behörden sowie eine fundierte schriftliche Zustimmung der Spender (oder in der Regel seine Eltern, zu erhalten, da Gewebe wird in der Regel von minderjährigen erhalten Kinder). Darüber hinaus sollten alle menschlichen Gewebe als potenziell infektiös und sollten geeignete Maßnahmen ergriffen werden, wie die Arbeit mit Handschuhen, etc. behandelt werden.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and Materials</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>PAA</td>
			<td>E15-842</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS</td>
			<td>PAA</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum-Gold</td>
			<td>PAA</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>PAA</td>
			<td>K41-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10 103 586 001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I, grade II bovine pancreatic</td>
			<td>Roche</td>
			<td>10 104 159 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 10x in PBS</td>
			<td>PAA</td>
			<td>L11-001</td>
			<td>stock conc. 20 mg/ml</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Protein Labelling kit</td>
			<td>Molecular Probes</td>
			<td>A-10235</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human CDR2 (purified)</td>
			<td>Bruno Kyewski, DKFZ- Heidelberg, Germany</td>
			<td></td>
			<td>labeled with Alexa Fluor 488</td>
		</tr>
		<tr>
			<td>anti-human CD45 (Pacific Blue)</td>
			<td>Biolegend</td>
			<td>304022</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human EpCAM (APC)</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-254</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-human CD11c (PE)</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-411</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45 Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-045-801</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>for tissue dissociator</td>
		</tr>
		<tr>
			<td>Percoll (density 1.130 g/ml)</td>
			<td>GE Healthcare, Life Sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile distilled Water (DNAse/ RNAse free)</td>
			<td>GIBCO</td>
			<td>10977-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Gamunex 10%</td>
			<td>Tajecris-Biotherapeutics</td>
			<td>G120052</td>
			<td>1:10 pre-dilution, use 20 &#956;l/1 x 106cells</td>
		</tr>
		<tr>
			<td>0.22 &#956;m filter</td>
			<td>Millex GS</td>
			<td>SLGS033SS</td>
			<td>Syringe driven</td>
		</tr>
		<tr>
			<td>Stericup filter unit</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td>Pump driven</td>
		</tr>
		<tr>
			<td>50 ml PC oak ridge centrifuge tubes</td>
			<td>Nalgene</td>
			<td>3118-0050</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>50 ml PP conical tubes</td>
			<td>Becton Dickinson</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm 5 ml test tubes</td>
			<td>Becton Dickinson</td>
			<td>352058</td>
			<td>FACS stainings</td>
		</tr>
		<tr>
			<td>Cell strainer 70 &#956;m</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>INSTRUMENTS</td>
		</tr>
		<tr>
			<td>Flow Cytometer-Sorter (BD FACSAriaTMIIu)</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Evolution R6 (rotor)</td>
			<td>Kendro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator REAX 2</td>
			<td>Heidolph</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093 235</td>
			<td>tissue dissociator</td>
		</tr>
	</tbody>
</table>

isolation of myeloid dendritic cells and epithelial cells from human thymus

In diesem Protokoll wird ein Verfahren, um dendritische Zellen (DC) und Epithelzellen (TEC) von dem menschlichen Thymus isolieren. DC und TEC sind die wichtigsten Antigen-präsentierenden Zellen (APC)-Typen in einem normalen Thymus gefunden, und es ist bekannt, dass sie unterschiedliche Rollen während Thymus-Auswahl zu spielen. Diese Zellen werden in unterschiedlichen Mikroumgebungen im Thymus lokalisiert und jeweils APC Typ macht nur einen geringen Population von Zellen. Die Biologie dieser Zelltypen weiter zu verstehen, ist die Charakterisierung dieser Zellpopulationen sehr wünschenswert, aber wegen ihrer niedrigen Frequenz, die Isolierung von jeder dieser Zelltypen erfordert ein effizientes und reproduzierbares Verfahren. Dieses Protokoll Details eine Methode für die Charakterisierung von verschiedenen zellulären Eigenschaften Zellen zu erhalten. Thymusgewebes mechanisch aufgeschlossen und nach verschiedenen Schritten des enzymatischen Verdau wird das erhaltene Zellsuspension mit einem Zentrifugationsschritt Percoll-Dichte angereichert. Für die Isolierung von myeloischen DC (CD11c &lt;sup&gt; +)-Zellen von geringer Dichte Fraktion (LDF) durch magnetische Zellsortierung immunoselected. Anreicherung von TEC Populationen (mTEC, cTEC) durch Verarmung der hämatopoetischen (CD45 hallo)-Zellen von geringer Dichte Percoll Zellfraktion wodurch ihre anschließende Trennung mittels Fluoreszenz-aktivierter Zellsortierung (FACS) unter Verwendung spezifischer Zellmarker erreicht. Für verschiedene Downstream-Anwendungen können die isolierten Zellen verwendet werden.

Als Ausgangsmaterial in diesem Protokoll verwenden wir Thymusgewebe von Kindern unterziehen Korrektur kardiovaskulären Chirurgie (Abteilung für Thorax-und Kardiovaskuläre Chirurgie, Universitätsklinikum Tübingen) nach der Einwilligung und unter institutionellen Richtlinien erhalten entfernt. Dieses Material kann verworfen stark in der Größe von 2 bis 30 g oder mehr variieren. Die Anzahl von MDC und TEC Teilmengen (cTEC und mTEC), die erhalten werden, hängt von der Größe als auch vom Alter des Thymus Gewebeprob...

Watch this Scientific Journal Video about Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus at JoVE.com

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Diese Protokollinformationen ein Verfahren zur Isolierung von Antigen-präsentierenden Zellen menschlichen Thymus über verschiedene Schritte der enzymatischen Verdauung des Gewebes, gefolgt von Dichtegradientenzentrifugation der Einzelzellsuspension und schließlich magnetisch und / oder FACS-Sortierung von Zellpopulationen von Interesse.

Isolierung von myeloider dendritischer Zellen und Epithelzellen Menschliche Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

isolation-myeloid-dendritic-cells-epithelial-cells-from-human

Research

JoVE Journal

Immunology and Infection

19.1K Aufrufe.  Hertie Institute for Clinical Brain Research.  Diese Protokollinformationen ein Verfahren zur Isolierung von Antigen-präsentierenden Zellen menschlichen Thymus über verschiedene Schritte der enzymatischen Verdauung des Gewebes, gefolgt von Dichtegradientenzentrifugation der Einzelzellsuspension und schließlich magnetisch und / oder FACS-Sortierung von Zellpopulationen von Interesse. 

Serie: Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolierung von Maus Lung Dendritischen Zellen

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Forschung

Immunologie und Infektion

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Isolierung und Charakterisierung von dendritischen Zellen und Makrophagen aus dem Mausdarm

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Isolierung und Immunfärbung Lymphozyten und dendritische Zellen aus Murine Peyer-Plaques

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Beurteilung der Innate Sensing von HIV-1-infizierten CD4<sup&gt; +</sup&gt; T-Zellen durch Plasmazytoide Dendritische Zellen unter Verwendung eines<em&gt; Ex-vivo-</em&gt; Co-Kultursystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T-Zellen erfassen Bakterien durch Transinfection von dendritischen Zellen

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Herstellung von menschlichen Monozyten abgeleiteten Dendritischen Zellen aus Vollblut

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Isolation von dendritischen Zellen aus menschlichen weiblichen Fortpflanzungsorgane für phänotypische und funktionelle Studien

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Entwicklung und Validierung eines empfindsamen Einzelmolekül Array digitale Enzym-linked Immunosorbentprobe Assay für menschliche Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.