Nous sommes reconnaissants à MM. Markus Manz et Irving Weissman pour fournir des réactifs. Nous reconnaissons également le service fourni par la cytométrie en flux et l&#39;analyse cellulaire tri de base Fonds de la première et deuxième laboratoire de base à la National Taiwan University College de l&#39;hôpital de médecine et NTU, respectivement. Ce travail a été soutenu par le Ministère de la Science et de la Technologie, Taiwan (MOST 102-2320-B-002-030-MY3) et les Instituts nationaux de recherche en santé, Taiwan (INDH-EX102-10256SI et INDH-EX103-10256SI).

/ 6 souris de type sauvage C57BL ont été achetées au Laboratoire national Animal Center (NLAC), Taiwan. Toutes les souris ont été élevées et maintenues dans des conditions exemptes d&#39;agents pathogènes spécifiques. Protocoles et des procédures d&#39;utilisation des animaux ont été examinés et approuvés par le soin et l&#39;utilisation des animaux dans les institutions Comité National Taiwan University College of Medicine (Numéro de permis: 20120075). En outre, les chercheurs ont fait tous les efforts pour réduire le potentiel de la douleur, de la souffrance ou de la détresse chez les animaux tout en effectuant des expériences. Toutes les procédures décrites ont été effectuées à la température ambiante, tout en portant des gants. 1. Préparation de l&#39;AC-6 cellules nourricières Remarque: L&#39;AC-6.21 (AC-6 en court) ligne de cellules stromales 18 (fourni par I. Weissman, l&#39;Université de Stanford) doit être maintenue dans un milieu RPMI complété avec du sérum fœtal bovin inactivé à la chaleur 15% (FBS). Pour servir de cellules nourricières, AC-6 cellules sont γ-irradié avec 3000 rad (30 Gy) un jour avant la co-culture pour empêcher leur prolifération. Remarque èmeAC-6 cellules ont tendance à perdre leur capacité de différenciation de soutien si surpeuplé gauche lors de l&#39;entretien, ou si leur numéro de passage est de plus de 20. <ol><li> Laver AC-6 cellules une fois avec 1 ml de solution saline tamponnée au phosphate de Dulbecco (DPBS) et retirer DPBS par aspiration. Traiter AC-6 cellules avec 0,8 ml de solution de trypsine (0,05% de trypsine et 0,5 mM d&#39;EDTA dans du DPBS) pour 3-5 minutes à 37 ° C, 5% de CO 2, puis arrêter la réaction par dilution avec 10 volumes de milieu de culture à faire suspension cellulaire unique. </li><li> Semences AC-6 dans des cellules 5.9x10 4 cellules / puits dans des plaques à 12 puits et incuber à 37 ° C, 5% CO 2 H / N. Note: La densité de l&#39;AC-6 cellules est très important car une densité inférieure favorisera génération CDC. Ensemencement à une densité de 5.9x10 4 cellules / puits permettront AC-6 pour atteindre la confluence le lendemain, ce qui est optimal pour la dérivation des PDC du CLP. </li><li> Irradier AC-6 cellules à 3000 rad (30 Gy) en utilisant γ-irradiation. Note: La dose utilisée pour irradier AC-6 est optimisé pour empêcher les cellules de proliférer et de les garder viable assez longtemps pour fournir les cytokines et contact cellule-cellule nécessaire à la différenciation des PED de CLPs encore. </li><li> Aspirer le milieu et le remplacer avec 1 ml de RPMI complet (RPMI avec 10% de FBS inactivé à la chaleur, 50 pg / ml de gentamycine et 50 uM de β-mercaptoéthanol). </li></ol> 2. Isolat de cellules BM de souris <ol><li> Sacrifiez souris par asphyxie au CO2 et dislocation cervicale. Placez les souris sur une plaque de dissection et stériliser avec 70% d&#39;éthanol. Faire une incision au milieu de l&#39;abdomen et enlevez la peau de la partie distale de la souris, y compris la peau qui recouvre les extrémités inférieures. </li><li> Disséquer les souris dans une hotte semi-stérile. Pour libérer le fémur et le tibia, le fémur le clip ci-dessus et ci-dessous tibias l&#39;articulation du genou. Détachez les muscles des os à l&#39;aide de ciseaux et placer les os dans une boîte de Pétri de 6 cm contenant 5 ml RPMI complet. </li><li> Déplacer vers une hotte de culture stérile. Remplir une seringue de 3 ml relié à une aiguille 27G avec RPMI complet. Coupez les deux extrémités des os à l&#39;aide de ciseaux, et insérer l&#39;aiguille de 27G pour rincer la moelle des os en injectant doucement RPMI complet une fois à chaque extrémité. </li><li> Préparer une suspension cellulaire unique par pipetage doux les cellules de haut en bas 3-5 fois dans la boîte de culture en utilisant la seringue sans aiguille. </li><li> Centrifuger les cellules pendant 5 minutes à 500 xg et décanter le surnageant. </li><li> Lyse des globules rouges avec une ml de tampon ACK (150 mM de NH 4 Cl, 10 mM de KHCO3, EDTA 0,1 mM) l&#39;incubation pendant 1 min et arrêt de la réaction par dilution avec 10 volumes de milieu RPMI complet. Laisser les cellules reposer pendant 5 min pour sédimenter bas amas de cellules mortes et les débris de tissu par gravité. Note: Ne pas se tenir plus de 5 minutes que cela se traduira par la perte de cellules due à régler de cellules viables. </li><li> Lentement décantation surnageant contenant les cellules à un nouveau tube de débris laissantderrière dans le tube d&#39;origine. </li><li> Centrifuger pendant 5 min à 500 g pour sédimenter les cellules, puis retirer et jeter le surnageant. </li><li> Reprendre cellules totales BM (typiquement 4-6 x 10 7 BM cellules sont obtenues à partir d&#39;une souris) dans 100 pi de tampon FACS (1x PBS + 2% de FBS + EDTA 1 mM). </li></ol> 3. Analyse FACS et le tri des PLC <ol><li> Ajouter anti-CD16 / 32 (clone hybridome 2.4G2 supernant, 50 ul / réaction ou 2.1 ug / réaction) dans des cellules dans un tampon FACS (étape 2.9) pendant 1-2 min afin de bloquer les récepteurs Fc. Remarque: anti-CD16 / 32 est également appelé bloc Fc, qui est ajouté pour empêcher une liaison non spécifique d&#39;anticorps à des cellules exprimant des récepteurs Fc, y compris les granulocytes, les monocytes et les lymphocytes B. </li><li> Cellules simultanément tacher avec les anticorps fluorescents suivants marqués par un colorant (pour 4x10 7 cellules) pendant 15 min sur la glace, en évitant la lumière ambiante. Anticorps: marqueurs de la lignée PE-conjugués (0,2 ug chacun) y compris anti-CD3 (17A2), anti-CD8 (53 à 6,7), anti-B220 (RA3-6B2), anti-CD19 (eBio1D3), anti-CD11b (M1 / 70), anti-Gr-1 (RB6-8C5), anti- Thy1.1 (HIS51), anti-NK1.1 (PK136), anti-Ter119 (TER-119) et anti-MHC-II (NIMR-4), 0,2 ug anti-c-Kit PerCP-Cy5.5- (2B8), 1 ug anti-Sca-1-FITC (D7), 0,4 ug anti-de M-CSFR-APC (AFS98), et 0,2 ug anti-IL-7Rα-PE-Cy7 (A7R34). </li><li> Laver les cellules avec 3 ml de tampon FACS, et centrifuger pendant 5 min à 500 x g. </li><li> Remettre les cellules dans 300 pi de tampon FACS et les cellules de filtre à travers un tamis cellulaire de 40 pm. </li><li> Laver le tube avec un tampon supplémentaire de 100 pi FACS pour récupérer toutes les cellules restantes et filtrer dans un FACS stériles tri tube en utilisant le même tamis cellulaire. </li><li> Effectuer une analyse de cytométrie de flux immédiatement après la coloration en utilisant des filtres et des tensions appropriées pour la détection du signal. Utilisez 488 nm laser pour détecter les anticorps FITC, PerCP-Cy5.5 marqués, 561 nm laser pour détecter la PE- et des anticorps PE-Cy7-labelded et 633 nm lasER pour détecter l&#39;anticorps marqué APC. </li><li> Trier PLC en fonction des marqueurs suivants lin c-kit - Sca-1 int int M-CSFR IL-7Rα - + (comme dans l&#39;étape 3.2 tachée) en utilisant un trieur de cellules. Recueillir les cellules triées dans un tube de 15 ml contenant 8 ml de RPMI complet comme un coussin. </li><li> Notez le nombre absolu de cellules triées comme indiqué par le trieur de cellules à la fin de la course. Typiquement, 5x10 4 PLC peuvent être obtenus à partir de la BM de souris une. </li></ol> 4. coculture CLP et AC-6 <ol><li> Centrifuger les cellules triées de l&#39;étape 3.7 pendant 10 min à 500 g, éliminer le surnageant et remettre le culot cellulaire avec assez de RPMI complet pour obtenir une densité cellulaire d&#39;environ 5x10 4 cellules / ml. Seed 500 cellules / puits dans la plaque de 12 puits contenant des cellules nourricières préparés à l&#39;étape 1.4. </li><li> Ajouter 100 ng / ml FL 19 (hu-Flt3L-Ig généré en interne en utilisant un système d&#39;expression fournie par M. Manz, UniversiHôpital ty Zürich, Suisse) et incuber à 37 ° C, 5% de CO 2 avec un contrôle visuel périodique de développement continu sous un microscope. Remarque: disponible dans le commerce FL humaine recombinante ou la souris FL peuvent être utilisés comme un substitut pour hu-Flt3L-Ig pour soutenir le développement continu. </li><li> Recueillir les cellules dans le surnageant à 12 jours et laver les puits une fois avec 0,5 ml de milieu RPMI compléter combinant les surnageants résultants. Ajouter 0,5 ml de milieu frais et débris les cellules adhérentes avec un racleur de cellules. </li><li> Moissonneuse le milieu contenant les cellules à partir des deux parties et centrifuger pendant 5 min à 500 x g. </li><li> Cellules surnageant et remettre Décanter dans un tampon FACS de 50 pi. Ajouter 50 ul anti-CD16 / 32 surnageant d&#39;hybridome et incuber pendant 1-2 min pour bloquer les récepteurs Fc. </li><li> Énumérer et colorer toutes les cellules avec les anticorps suivants (0,05 ug chacun) anti-CD11c-APC (N418), anti-CD11b FITC-et anti-B220-PE. </li><li> Lavez et centrifuger les cellules que décrired à l&#39;étape 3.3. </li><li> Reprendre les cellules dans 100 pi de tampon FACS, porte sur CD11c + et d&#39;analyser pour CDCS (CD11c + CD11b + B220 -) et pDC (CD11c + CD11b - B220 +) 17. </li></ol>

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Authors have nothing to disclose.

Ici, nous décrivons un système in vitro en culture pour la génération robuste des pays en développement, et en particulier les pDC, à partir d&#39;un petit nombre de CLP. Le caractère unique de ce système de culture est due à l&#39;utilisation de l&#39;AC-6, les cellules d&#39;une lignée cellulaire stromale, en tant que dispositifs d&#39;alimentation. Cette approche a été montré pour fournir non seulement les cytokines, telles que IL-7, SCF, M-CSF et FL 20, mais aussi le contact...

Les cellules dendritiques (CD) sont des cellules présentatrices d&#39;antigène professionnelles qui jouent un rôle important dans le contrôle des réponses immunitaires 1. Alors que les PED sont hétérogènes, ils peuvent être classés en deux populations, les CD classique (CDCS) et DCS plasmacytoïdes (pDC) 2,3. En plus des organes lymphoïdes, CDCS et pDC sont également présents dans les tissus y compris les poumons, l&#39;intestin et de la peau 2. La morphologie du CDCS et pDCs diffère, avec CDCS présentant projections de dendrites-like et la forme des PDC comme étant des cellules plus de plasma. En outre, le marqueur DC de la souris commune, CD11c, est plus fortement exprimé sur CDCS que sur les pDC. En outre, CDC peut encore être divisé en CD11b + CD24 - CDCS et CD11b - CD24 + CDCS, qui expriment toutes deux des niveaux plus élevés de CMH de classe II et molécules costimulatrices que font pDCs 2. PDCs matures, d&#39;autre part il a été démontré, pour exprimer sélectivement Siglec-H et BST2 4. Functionally, CDCS sont meilleures cellules présentatrices d&#39;antigène que sont pDCs; Toutefois, pDCs peuvent produire une grande quantité d&#39;interféron de type I lors de l&#39;infection par le virus ou la stimulation inflammatoire 5. Les deux CDCS et pDC sont de courte durée, et par conséquent, doivent être constamment reconstituées à partir de progéniteurs dans la moelle osseuse (BM) 6,7. Le transfert adoptif de progéniteurs myéloïdes communs (CMPS) et les progéniteurs lymphoïdes communs (cLPS) dans des souris mortellement irradié démontre que CDCS et pDCs peuvent être générés à partir de deux lignées 8-10. Cependant, il existe un sous-ensemble de pDC qui expriment RAG1 / 2 et possédant des segments réarrangés DJ au locus IgH 11,12. Ces cellules partagent aussi des similitudes moléculaires avec des lymphocytes B, y compris l&#39;expression du marqueur de B220, récepteurs de l&#39;acide détection nucléiques (TLR7 / TLR9), et les facteurs de transcription (SPIB et BCL11A) 13, dispose de tous croit être les signatures de la lignée lymphoïde. Par conséquent, CLPs peut être un bon choix pour la production in vitro de la lignée de pDC parce similitude. Bien que la fréquence des deux CDCS et pDC chez les humains et les souris est très faible 6, les DC, en particulier CDCS, peut être produite in vitro à partir de progéniteurs BM ou en présence de cytokines, telles que GM-CSF ou le ligand Flt3 11,14 (FL ) en utilisant des systèmes sans cellules nourricières 11,15,16. Malheureusement, il est impossible de produire un grand nombre de pDC in vitro utilisant FL 11,15,16. Auparavant, nous avons démontré que les pDC peuvent être générées de manière efficace in vitro de PLC au moyen du système d&#39;alimentation AC-17 6. L&#39;avantage d&#39;utiliser la lignée cellulaire stromale AC-6 dans le système de culture est que cela permet un contact cellule-cellule et sécrétion de cytokines qui favorisent la production d&#39;un grand nombre de pDC à partir de PLC. Bien que la production de ce système est très robuste, la réplication attentif des procédures décrites ci-dessous est nécessaire in Afin d&#39;assurer la production efficace des PDC.

<table>
	<tbody>
		<tr>
			<td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td>
			<td>Biolegend</td>
			<td>108408</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse NK1.1 (PE), clone PK136</td>
			<td>Biolegend</td>
			<td>108708</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse&#160; CD11b (PE), cloneM1/70</td>
			<td>Biolegend</td>
			<td>101208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD19 (PE), clone eBio1D3</td>
			<td>Biolegend</td>
			<td>115508</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse B220 (PE), clone RA3-6B2</td>
			<td>Biolegend</td>
			<td>103208</td>
			<td>linage marker/FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD3 (PE), clone 17A2</td>
			<td>Biolegend</td>
			<td>100308</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a (PE), clone 53-6.7</td>
			<td>Biolegend</td>
			<td>100707</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse MHC-II (PE), clone NIMR-4</td>
			<td>Biolegend</td>
			<td>107608</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119 (PE), clone TER-119</td>
			<td>Biolegend</td>
			<td>116208</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse Thy1.1 (PE), clone HIS51</td>
			<td>eBioscience</td>
			<td>12-0900-83</td>
			<td>linage marker</td>
		</tr>
		<tr>
			<td>Anti-mouse M-CSFR (APC), clone AFS98</td>
			<td>Biolegend</td>
			<td>135510</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td>
			<td>Biolegend</td>
			<td>105824</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse Sca-1 (FITC), clone D7</td>
			<td>Biolegend</td>
			<td>108106</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td>
			<td>Biolegend</td>
			<td>135014</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td>
			<td>Biolegend</td>
			<td>117328</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (FITC), clone M1/70</td>
			<td>Biolegend</td>
			<td>101206</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
		<tr>
			<td>FACS sorting tube&#160;</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo LLC</td>
			<td></td>
			<td>Flow analysis sofrware</td>
		</tr>
	</tbody>
</table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

Les cellules dendritiques plasmacytoïdes (pDC) sont de type I puissant interféron (IFN-I) des cellules productrices qui sont activés en réponse à une infection ou au cours des réactions inflammatoires. Malheureusement, l&#39;étude de la fonction PDC est entravé par leur faible fréquence dans les organes lymphoïdes et les méthodes existantes pour vitro génération DC favorise principalement la production des CDC sur les pDC. Ici, nous présentons une approche unique pour générer efficacement des pDC de progéniteurs lymphoïdes communs (cLPS) in vitro. Plus précisément, le protocole décrit les détails comment purifier CLPs de la moelle osseuse et de générer des pDC par co-culture avec AC-6 cellules nourricières gamma-irradiées en présence de ligand Flt3. Une caractéristique unique de ce système de culture est que les PLC migrent au-dessous des cellules AC-6 et deviennent des cellules de la zone formant pavées, une étape cruciale pour l&#39;expansion pDC. PED morphologiquement distinctes, à savoir PDC et les CDC, ont été générés après environ deux semaines avec une composition de 70-90% pDC dans des conditions optimales. En règle générale, le nombre de pDC générés par ce procédé est d&#39;environ 100 fois du nombre de têtes de série PLC. Par conséquent, cela est un nouveau système avec lequel pour générer vigoureusement le grand nombre de pDC nécessaires pour faciliter d&#39;autres études sur le développement et la fonction de ces cellules.

Un total de 7 4-6x10 cellules BM sont généralement isolé de fémurs et tibias d&#39;un 6-8 sem-vieux, de type sauvage C57BL / 6 souris. Pour trier CLP, cellules totales BM sont colorées avec des anticorps PE-conjugué contre des marqueurs de la lignée (CD3, CD8, B220, CD19, CD11b, GR-1, Thy1.1, NK1.1, Ter119, et CMH-II), Anti -c-Kit-PerCP / Cy5.5, anti-Sca-1-FITC, anti-de M-CSFR et APC-anti-IL-7Rα-PE / Cy7, analysées et triées avec un trieur de cellules. La stratégie de tri pour CLP est représenté ...

Watch this Scientific Journal Video about In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System at JoVE.com

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

In Vitro génération de cellules dendritiques plasmacytoïdes murin de Common lymphoïde progéniteurs utilisant le système de chargeur AC-6

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

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Research

JoVE Journal

Immunology and Infection

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

8.3K vues.  National Taiwan University College of Medicine. In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand. 

Vidéo: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Génération et d&#39;étiquetage des murins de la moelle osseuse des cellules dendritiques dérivées des nanocristaux Qdot pour les études de suivi

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Recherche

Immunologie et infection

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 ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Génération d’un nouveau vaccin à cellules dendritiques utilisant des cellules souches de mélanome et de cancer malpighien

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Évaluer le développement de cellules murines dendritiques plasmacytoïdes dans les plaques de Peyer Utiliser le transfert adoptif de progéniteurs hématopoïétiques

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

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 ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Génération de Immature, d&#39;âge mûr et tolérogène cellules dendritiques avec Metabolic Différentes phénotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Génération et identification de GM-CSF dérivé alvéolaires comme Macrophages et cellules dendritiques à partir de moelle osseuse de souris

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-tri cellulaire activé pour la purification de cellules dendritiques plasmacytoïdes de la moelle osseuse de souris

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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 ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nouveau système exempt d’alimentation pour la Production de masse des cellules tueuses naturelles murins In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...