Nous sommes reconnaissants envers les chirurgiens du département de chirurgie thoracique et cardiovasculaire, Clinique universitaire de Tübingen pour nous fournir les échantillons de thymus et Bruno Kyewski (DKFZ, Heidelberg, Allemagne) pour fournir l&#39;anticorps CDR2. Nous tenons également à remercier Hans-Jörg Bühring et Sabrina Grimm du centre de tri (Université de Tübingen). Ce travail a été soutenu par le SFB 685 et la Fondation Hertie.

<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>

Aucun conflit d&#39;intérêt déclaré.

Le protocole décrit ici est une modification du protocole publié par Gotter et al 4. Les étapes critiques dans le protocole sont la condition et la préparation initiale du tissu ainsi que la séparation de densité Percoll. Nous vous recommandons fortement de traiter le tissu le plus tôt possible après la collecte. Il est important de travailler vite mais bien lors du nettoyage et couper le tissu. Lors du lavage des thymocytes décrit à l&#39;étape 2.3, il est essentiel de trouver le juste é...

Le thymus est l&#39;organe dans lequel le développement des cellules T se produit. Sa taille relative et absolue diminue avec l&#39;âge quand il est remplacé successivement par de la graisse bien que l&#39;activité du thymus peut encore être détecté dans la vieillesse. Son importance pour la réponse immunitaire a été démontrée dans le début des années 1960 1. Le répertoire des cellules T est façonnée par l&#39;interaction des récepteurs des cellules T avec des complexes peptide-MHC sur les différents types de thymus APC, qui assurent la survie ou la mort des indices de développement de cellules T, ce qui entraîne un répertoire fonctionnel et largement auto-tolérance des lymphocytes T 2. Environ 98% des cellules dans le thymus humain développent des lymphocytes T appelés thymocytes. Les 2% restants se composent d&#39;un certain nombre de différents types de cellules, y compris une variété de TEC (corticale, médullaire, sous-capsulaire), myéloïde et CD plasmacytoïdes (MDC, PDC), les macrophages, les lymphocytes B, les cellules matures re-circulation T, les granulocytes, faibroblasts, les cellules endothéliales et les cellules épithéliales de très rares avec un phénotype d&#39;expression qui ressemble à celle des cellules provenant d&#39;autres tissus tels que les muscles, les neurones et de l&#39;épithélium des voies respiratoires (Figure 1). Parmi ceux-ci, TEC et DC sont les principaux types d&#39;APC présents dans un thymus normal. Au cours des dernières années, la purification de ces types d&#39;APC pour la culture et le profilage moléculaire a gagné de plus en plus d&#39;intérêt. En raison de leur basse fréquence, l&#39;isolement de l&#39;un de ces types de cellules pour une analyse détaillée nécessite une procédure efficace, reproductible et économique. La méthode présentée ici est une modification d&#39;études publiées antérieurement 3,4. Comme avec n&#39;importe quel autre tissu, l&#39;extraction des cellules du thymus peut être obtenue en décomposant par voie enzymatique de la cellule-cellule et les réseaux d&#39;interactions cellule-matrice, pour obtenir une suspension de cellules uniques. Il existe certains paramètres comme la bonne efficacité de dissociation, le rendement cellulaire, la viabilité des cellules et le maintien des cellules smarqueurs de urface qui sont cruciales et doivent être optimisées pour l&#39;isolement réussi de ces populations de cellules rares. Dans ce protocole, l&#39;isolement des sous-ensembles de DC et TEC est réalisée par formation d&#39;une suspension à cellule unique du tissu par rupture mécanique et la digestion enzymatique. Nous utilisons la collagénase A de Clostridium histolyticum, qui a un rapport équilibré des différentes activités enzymatiques, à briser le collagène natif qui détient le tissu ensemble. DNase I est inclus dans la solution enzymatique pour réduire l&#39;agrégation cellulaire en raison de l&#39;ADN libre à partir de cellules mortes (thymocytes sont très sensibles). Nous fournissons également une approche alternative à la digestion du tissu enzymatique typique impliquant un traitement de tissu mécanique et enzymatique assistée par un dissociateur de tissu. La suspension cellulaire unique est ensuite soumis à une centrifugation unique de densité Percoll pour l&#39;enrichissement de la fraction de faible densité (LDF) de cellules. A partir de cette fraction de cellules, DC peut être isolé par coloration fou marqueurs DC-surface (c.-à-CD11c +) et en utilisant une séparation magnétique ou une cellule activé par fluorescence (FACS). A la différence des cellules lymphoïdes comprenant la grande majorité des cellules dans le thymus, les TEC n&#39;expriment pas CD45 à des niveaux élevés, mais sont positifs pour le adhésion cellulaire épithéliale molécule EpCAM. Ctec peut être distingué du TEC médullaire par l&#39;expression d&#39;un antigène reconnu non encore défini par le CDR-2 (cortical dendritique réticulocytes-2) un anticorps de 4,5 et l&#39;expression d&#39;EpCAM un peu plus faible. L&#39;écart-co-expression de EpCAM et CDR2 permet l&#39;isolement efficace de ces sous-ensembles de TEC par l&#39;intermédiaire de la cellule à grande vitesse de tri 6. Le protocole présenté ici est optimisée pour les tissus du thymus humain. La durée de la procédure dépend de la quantité de tissu et de la capacité de l&#39;expérimentateur, ainsi que la vitesse du trieur de cellules, si tri FACS est utilisé. Normalement, le protocole pour l&#39;isolement de CC peut être achevée dans un délai de 5-6 H et à l&#39;isolement des TEC en 8-10 heures. L&#39;isolement des DC et des sous-ensembles de TEC de tissu thymique est sensible au temps. Plus la procédure d&#39;isolement, meilleure est l&#39;état des cellules. Enfin, les cellules isolées peuvent être utilisées pour d&#39;autres investigations, comme des études comparatives de l&#39;ARNm et l&#39;expression des protéines, des expériences de PCR, l&#39;isolement de la protéine, le profilage moléculaire (à savoir les transcriptomique, micro-analyse de l&#39;ARN), ainsi que la culture de la cellule 6. Déclaration d&#39;éthique Afin d&#39;être en mesure de travailler avec le tissu de thymus humain, le chercheur doit obtenir l&#39;approbation du comité d&#39;éthique local ou les autorités responsables ainsi que d&#39;un consentement écrit du donneur (ou habituellement ses parents, puisque le tissu est généralement obtenu à partir de mineurs les enfants). En outre, tous les tissus humains doivent être traités comme étant potentiellement infectieux et des mesures appropriées doivent être prises, telles que le travail avec des gants, etc.

<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

Dans ce protocole, nous fournissons un procédé pour isoler des cellules dendritiques (DC) et les cellules épithéliales (TEC) à partir du thymus humain. DC et TEC sont la principale cellule présentatrice d&#39;antigène (APC) de types trouvés dans un thymus normal et il est bien établi que ils jouent des rôles distincts lors de la sélection thymique. Ces cellules sont localisées dans des micro-environnements distincts dans le thymus et chaque type APC ne constitue qu&#39;une petite population de cellules. Pour mieux comprendre la biologie de ces types de cellules, la caractérisation de ces populations de cellules est très souhaitable, mais en raison de leur basse fréquence, l&#39;isolement de l&#39;un de ces types de cellules nécessite une procédure efficace et reproductible. Ce protocole détaille une méthode pour obtenir des cellules appropriées pour la caractérisation des propriétés cellulaires divers. Tissu thymique est mécaniquement perturbé et après différentes étapes de digestion enzymatique, la suspension de cellules résultante est enrichi à l&#39;aide d&#39;une étape de centrifugation par densité de Percoll. Pour l&#39;isolement des myéloïde DC (CD11c &lt;sup&gt; +), des cellules provenant de la fraction de faible densité (LDF) sont immunosélectionné par tri cellulaire magnétique. Enrichissement des populations de TEC (mTEC, la technologie cTEC) est réalisé par déplétion des hématopoïétiques (CD45 hi) des cellules de la fraction cellulaire de faible densité Percoll permettant leur isolement subséquent par tri cellulaire activé par fluorescence (FACS) en utilisant des marqueurs cellulaires spécifiques. Les cellules isolées peuvent être utilisées pour différentes applications en aval.

Comme matière première dans ce protocole, nous utilisons des tissus thymus enlevé des enfants subissant une chirurgie cardiovasculaire correctives (ministère de la chirurgie thoracique et cardiovasculaire, Clinique universitaire de Tübingen) obtenus après consentement éclairé et sous les directives institutionnelles. Ce produit rejeté peut varier considérablement en taille 2-30 g ou plus. Le nombre de MDC et des sous-ensembles de TEC (technologie cTEC et MTEC) qui sont obtenus dépend de la taille ainsi que l&...

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

Ce protocole décrit en détail une méthode pour isoler des cellules présentant l&#39;antigène à partir de thymus humain par l&#39;intermédiaire de différentes étapes de digestion enzymatique du tissu suivie d&#39;une centrifugation de densité de la suspension de cellules individuelles et enfin le tri magnétique et / ou FACS des populations de cellules d&#39;intérêt.

Isolement des cellules dendritiques myéloïdes et les cellules épithéliales du thymus humain

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 vues.  Hertie Institute for Clinical Brain Research.  Ce protocole décrit en détail une méthode pour isoler des cellules présentant l'antigène à partir de thymus humain par l'intermédiaire de différentes étapes de digestion enzymatique du tissu suivie d'une centrifugation de densité de la suspension de cellules individuelles et enfin le tri magnétique et / ou FACS des populations de cellules d'intérêt. 

Vidéo: 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.

L&#39;isolement des cellules de souris poumon dendritiques

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 ...

Recherche

Immunologie et infection

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.

L&#39;isolement et la caractérisation des cellules dendritiques et les macrophages de l&#39;intestin de souris

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.

Et isoler immunocoloration des lymphocytes et des cellules dendritiques à partir des plaques de Peyer murin de

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.

L&#39;évaluation de l&#39;Innate détection du VIH-1 CD4 infectés<sup&gt; +</sup&gt; Cellules T par les cellules dendritiques plasmacytoïdes Utilisation d&#39;un<em&gt; Ex vivo</em&gt; Système de co-culture.

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.

Isolement, identification et la purification de cellules murines thymique épithéliales

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.

Cellules T Capturez bactéries par Transinfection de cellules dendritiques

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.

Génération de cellules humaines dérivées de monocytes dendritiques du sang total

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.

Isolement de cellules dendritiques de l’appareil reproducteur féminin humain pour des études phénotypiques et fonctionnelles

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.

Développement et Validation d’une seule molécule ultrasensible de tableau numérique Enzyme-linked Immunosorbent Assay for Human interféron-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.