The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

Dans cette publication vidéo et de protocole, toutes les procédures d&#39;animaux ont été réalisées conformément au protocole approuvé pour les animaux par cantonal Autorité de Bâle-Ville, Suisse. 1. ganglions lymphatiques Préparation et digestion <ol><li> L&#39;eau de pré-chauffage dans un bêcher à 37 ° C sur un agitateur magnétique à plaque chauffante. </li><li> Préparer de base moyen de la manière suivante: milieu DMEM (sans pyruvate) supplémenté avec 2% de FCS, 1,2 mM de CaCl2 et Pen / Strep (100 unités de pénicilline, 100 ug de streptomycine). </li><li> Stériliser tous les instruments de dissection avant utilisation. </li><li> Euthanasier souris ganglionnaire donateurs par CO 2 asphyxie et disséquer de façon aseptique les ganglions lymphatiques. Ne pas disséquer la graisse entourant. NOTE: Ce protocole est optimisé pour le noeud périphérique drainage lymphatique peau (inguinale, brachiale, axillaire). </li><li> Placez les ganglions lymphatiques dans une boîte de Pétri stérile contenant 2 ml de glace milieu basique froid. </li><li> Perturber le ganglion lymphatique capsule l&#39;aide de deux aiguilles de 25 G fixés sur seringue de 1 ml. </li><li> Transférer le tissu des ganglions lymphatiques perturbé dans un tube à fond rond de 5 ml contenant 750 polypropylène milieu de base supplémenté avec ul de 1 mg / ml de collagénase IV et 40 ug / ml de DNAse I. </li><li> Ajouter un agitateur magnétique stérile dans chaque tube. </li><li> Placer le tube dans le bécher avec 37 ° C préchauffé eau et remuer les tubes à une vitesse lente (1 tour / s) pendant 30 min. </li><li> Retirer le tube de l&#39;agitateur magnétique avec plaque chauffante et laisser des fragments de ganglions lymphatiques s&#39;installent. </li><li> Retirez délicatement le surnageant enrichi en &quot;cellule non-stromal&quot;. REMARQUE: Si l&#39;analyse de T, B, les cellules dendritiques et CD45 - CD31 - gp38 - est prévu, sauf la &quot;cellules stromales non&quot; fraction. </li><li> Laver le tissu des ganglions lymphatiques restant une fois avec 750 pi de milieu basique. REMARQUE: Cette étape est nécessaire uniquement si vous travaillez avec des souris immuno-compétentes. </li><li> Laissez fragments de ganglions lymphatiques s&#39;installent. </li><li> Retirez lefraction flottante des cellules non-stroma. </li><li> Ajouter aux fragments de ganglion lymphatique 750 pi de milieu de base supplémenté avec 3,5 mg / ml de collagénase D et 40 pg / ml de DNase I. </li><li> Placer le tube de retour dans le bécher contenant 37 ° C préchauffé eau. </li><li> Digérer le tissu des ganglions lymphatiques de 5 min, tout en agitant lentement. </li><li> Lymphatiques fragments de tissus de noeud désagrégées par pipetage et mélange 700 ul de 10 cycles à la vitesse maximale à l&#39;aide d&#39;une pipette multicanaux automatisé. NOTE: Ce qui perturbe le tissu des ganglions lymphatiques pour améliorer la digestion. </li><li> Placer le tube de retour dans le bécher avec 37 ° C de l&#39;eau préchauffée. </li><li> Des fragments de tissu de ganglion lymphatique digérer pendant encore 10 minutes tout en remuant lentement. </li><li> Ventiler fragments de tissus de ganglions lymphatiques par pipetage et mélange pendant 99 cycles à vitesse maximale à l&#39;aide d&#39;une pipette multicanaux automatisé. </li><li> Ajouter 7,5 pl d&#39;EDTA 0,5 M pour assurer le maintien de la suspension de cellules isolées. </li><li> Des fragments de tissu de ganglion lymphatique Ventiler par tuyauPrép et en mélangeant pendant 99 cycles à vitesse maximale à l&#39;aide d&#39;une pipette multicanaux automatisé. </li><li> Ajouter 750 pi de milieu de base et passer les cellules à travers une maille de nylon de 70 um. </li><li> Centrifugeuse suspension cellulaire 5 min à 1500 xg, 4 ° C. </li><li> Les cellules stromales peuvent maintenant être utilisés pour une analyse ultérieure. </li></ol> 2. coloration des cellules des ganglions lymphatiques stromales <ol><li> Utilisez une combinaison de l&#39;anti-CD45, anti-podoplanin (gp38), anticorps anti-CD31 de reconnaître avec succès des cellules TRC, ESL, BEC et DN. </li><li> Incuber le tissu des ganglions lymphatiques digéré avec Live traqueur / Dead, anti-CD45-FITC (1: 200), anti-gp38-PE (1: 200), anti-CD31-APC (1: 200) dans 100 ul de HBSS contenant 2 % de FCS pendant au moins 20 min, 4 ° C, dans l&#39;obscurité. </li><li> Laver les cellules en ajoutant 500 l de HBSS contenant 2% de SVF </li><li> Centrifugeuse suspension cellulaire 3 min à 1500 xg, 4 ° C. </li><li> Re-suspendre dans 100 ul de HBSS contenant 2% de FCS. </li><li> Exécutez les cellules colorées dans un cytomètre de flux EQUIPPed avec les optiques suivantes: source d&#39;excitation avec un maximum de trois lasers: bleu (488 nm, refroidi par air, 20 mW à l&#39;état solide), rouge (633 nm, 17 mW HeNe) et le violet (405 nm, 30 mW à l&#39;état solide) . </li><li> Porte CD45 - cellules à exclure les cellules hématopoïétiques. </li><li> maillots Gate (FSC-W) et des cellules vivantes (ELS / trackers DEAD) à exclure doublets et les cellules mortes. </li><li> Terrain gp38 contre CD31 pour visualiser TRC (gp38 + CD31 -), ESL (gp38 + CD31 +), BEC (gp38 - CD31 +) des cellules (voir la figure 1) et DN (gp38 - - CD31). </li></ol>

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The authors declare they have no competing financial interests.

L&#39;étude des cellules stromales de ganglions lymphatiques est récemment devenu un axe de recherche en raison du développement de deux protocoles de digestion publiés 6,13. Les deux protocoles sont suffisants pour obtenir des cellules stromales noeud unique lymphatiques, mais diffèrent dans l&#39;utilisation d&#39;enzymes de digestion et le temps de digestion. Etant donné que les cellules stromales et les marqueurs de surface sont sensibles à la digestion enzymatique et une contrainte mécanique, un ...

Les ganglions lymphatiques sont des compartiments spécialisés où les réponses immunitaires adaptatives contre des antigènes étrangers autonomes et sont initiés et coordonnés. La procédure présentée ici décrit une digestion enzymatique courte combinée avec pipetage mécanique automatisé pour obtenir des ganglions lymphatiques suspension cellulaire unique et accéder aux cellules stromales ganglionnaires viables qui maintiennent l&#39;expression de surface de plusieurs molécules. former cellules stromales ganglionnaire l&#39;échafaud du ganglion lymphatique et remplit trois grandes fonctions: ils filtrent première fluides corporels pour déguster des antigènes, des agents pathogènes et leur pathogène motif moléculaire associé (PAMP), ainsi que des cytokines et danger motif moléculaire associé (atténue) présente dans le corps. Deuxièmement, ils attirent et instruisent les cellules présentatrices d&#39;antigènes (APC) et des lymphocytes d&#39;interagir et d&#39;initier des réponses immunitaires adaptatives; et la troisième, ils fournissent un environnement structurel pour l&#39;homéostasie et la différenciation des lymphocytes 1-3. Pendant les cellules ganglionnaires du stroma de l&#39;inflammation produisent des facteurs de croissance, des cytokines et des chimiokines, à adapter l&#39;organisation de gonflement ainsi l&#39;interaction entre les cellules dendritiques (DCs), T et les cellules B. L&#39;orchestration de réponses immunitaires est seulement possible en raison de l&#39;architecture structurale complexe formé par les différentes populations de cellules stromales. Les cellules stromales de ganglions lymphatiques sont des cellules CD45 négatives et peuvent être distinguées par l&#39;expression de CD31 ou de gp38 dans les cellules endothéliales et fibroblastiques 1 -6. Gp38 + CD31 - définit les cellules de la zone T réticulaire (TRC, également connu sous le FRC: les cellules réticulaires fibroblastiques), gp38 + CD31 + définit cellules lymphatiques endothéliales (LEC), gp38 - CD31 + définit les cellules endothéliales de sang (BEC). En outre, la caractérisation des sous-populations a révélé l&#39;existence d&#39;autres cellules stromales de ganglions lymphatiques. En effet, une petite population de cellules pericyte comme a été caractérisée dans legp38 - CD31 - population 7. Par conséquent, l&#39;adaptation de la procédure d&#39;isolement est avantageux pour l&#39;identification et la caractérisation des propriétés fonctionnelles différentes cellules stromales de ganglions lymphatiques. Avant le développement des ganglions lymphatiques cellules stromales digestion protocoles de l&#39;étude des cellules stromales de ganglion lymphatique a été limitée à des observations in situ en utilisant la section de tissu et la microscopie. Néanmoins, des études structurales et fonctionnelles ont montré des caractéristiques importantes des cellules stromales de ganglions lymphatiques. Les cellules stromales de ganglions lymphatiques sont associés à podoplanin, du collagène et de la matrice extracellulaire des protéines (ECM) pour former un complexe d&#39;une structure en 3 dimensions appelé système de conduites qui transporte les protéines lymphatiques et de masse moléculaire associé-bas à partir du sinus sous-capsulaire du ganglion de la haute endothéliale veinules dans la zone des cellules T 8. DCs sont en contact étroit avec les cellules du stroma et peut être observé dans la saillie tubulaire constructure duit à l&#39;échantillon liquide et de détecter les antigènes 8. L&#39;interaction des cellules stromales de ganglions lymphatiques (TRC et ESL) avec les PED est médiée par la presse et la présentation des chimiokines CCL21 et CCL19 9,10. CCL19 et CCL21 sont reconnus par le récepteur CCR7 faciliter les DC et les cellules T à migrer vers la zone des cellules T des ganglions lymphatiques 4,11. Malgré l&#39;utilisation de chimiokines similaires, les cellules T ont PED et les routes de migration différentes dans les ganglions lymphatiques 12. Par la suite, en utilisant une digestion enzymatique de ganglion lymphatique et de l&#39;isolement des cellules stromales de ganglions lymphatiques pures, des études fonctionnelles ont été effectuées sur le rôle des différentes cellules des ganglions lymphatiques de stroma et de leur capacité à interagir avec des DC et des lymphocytes T / B 6,13. Tout d&#39;abord, la diaphonie entre les cellules T productrices d&#39;IFN-effectrices de γ et de cellules stromales de ganglions lymphatiques induit la production d&#39;oxyde nitrique du métabolite montré à atténuer les réponses des lymphocytes T et la prolifération dans des organes lymphoïdes secondaires 14 à 16. Deuxièmement, la lymphe node cellules stromales ont été rapportés pour soutenir la différenciation de sous-ensembles de régulation à courant continu par l&#39;intermédiaire de la production d&#39;IL-10 17, et à moduler naïf homéostasie des cellules T par l&#39;intermédiaire de la production d&#39;IL-7 6,18. En troisième lieu, l&#39;expression des TLR dans les cellules stromales de ganglions lymphatiques suggère que les cellules stromales sont sensibles à un signal dérivé de l&#39;infection ou des auto-molécules libéré au cours de la lésion tissulaire. En effet, le traitement de cellules stromales de ganglions lymphatiques avec le ligand de TLR3 poly (I: C) induit une régulation à la hausse modeste de grande expression d&#39;histocompatibilité de classe complexe I et la régulation positive de co-inhibiteur molécule PD-L1, mais pas de molécules de costimulation, ce qui des changements dramatiques dans les tissus périphériques antigènes expression 19. Plusieurs groupes ont montré des cellules stromales de ganglions lymphatiques expriment des antigènes de tissus périphériques et induire une tolérance des cellules T autoréactives 19,21-27. Par conséquent, la compréhension des interactions entre les cellules stromales de ganglions lymphatiques et l&#39;autre re migratoire etcellules ganglionnaires sident aideront à trouver de nouvelles molécules cibles pour permettre l&#39;activation ou la suppression de la réponse immunitaire lors de l&#39;inflammation. Par conséquent, la mise en œuvre de la séparation enzymatique de la publication du ganglion lymphatique est nécessaire. Protocoles publiés précédemment utilisent différentes combinaisons de base de la collagénase-digestion enzymatique avec une faible contrainte mécanique 6,19,20. Cependant, de longues incubations avec des enzymes de digestion ou de la combinaison différente de digestion enzymatique peuvent dégrader diverses molécules de surface nécessaires pour analyser l&#39;état d&#39;activation et d&#39;identifier de nouvelles cellules stromales ganglionnaires. Selon le type de l&#39;analyse de cellules du stroma, le Protocole de liaison Protocole Fletcher ou peut-être plus approprié. Dans la procédure décrite, une digestion enzymatique légèrement plus courte est associée à la ventilation mécanique automatisé pour minimiser la dégradation de surface marqueur des cellules stromales nœud lymphatiques viable. Cette procédure permet d&#39;isoler hautement reproductible etdistinction des populations de cellules stromales de ganglions lymphatiques avec une faible variabilité et plus de 95% de viabilité. Les cellules stromales de ganglions lymphatiques fraîchement isolées peuvent être directement utilisés pour l&#39;expression de marqueurs de surface, l&#39;analyse des protéines, et des études de la transcription, ainsi que la création de lignées de cellules stromales pour effectuer des tests fonctionnels in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="959">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:231px;">LifeTechnologies-Gibco</td>
			<td style="width:119px;">41965-039</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td>Final concentration 2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">499609</td>
			<td>Final concentration 1.2 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Collagenase IV</td>
			<td style="width:231px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;"></td>
			<td>&#62;160 units per mg dry weight, use at final concentration of 1 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:231px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td>use at 3.5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNAse I</td>
			<td style="width:231px;">Roche&#160;</td>
			<td style="width:119px;">11284932001</td>
			<td>use at 40 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stiring magnets</td>
			<td style="width:231px;">FAUST</td>
			<td style="width:119px;"></td>
			<td>5 mm long-2 mm &#248;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polystyren Round-Bottom Tubes 5ml</td>
			<td style="width:231px;">Falcon-BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic stirrer with heating funktion</td>
			<td style="width:231px;">IKA-RCT-standard</td>
			<td style="width:119px;">9720250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petridishes 100 mm, sterile</td>
			<td style="width:231px;">TPP</td>
			<td style="width:119px;">6223201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needles</td>
			<td style="width:231px;">Terumo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD45 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD11c Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse Podoplanin Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 8.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD31 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone MEC13.3</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;"> 
			Eppendorf Xplorer plus, Multichannel</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:119px;">4861 000.821/830</td>
			<td>1.250 &#181;l max. volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD140a</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone APA5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD80</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 16-10A1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD40</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 1C10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse I-Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone AF6-120.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD274 (PD-L1)</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 10F.9G2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of murine lymph node stromal cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Le présent protocole est un protocole de modification de digestion publié par Link et al. 2007 6 avec un temps de digestion plus courte (45 minutes au maximum), en raison de la désagrégation mécanique avec une pipette à canaux multiples automatisé. En outre, la procédure est plus uniforme, minimise la dégradation de marqueurs de surface sur les différentes cellules du stroma de ganglion lymphatique et permet la manipulation de plus d&#39;un échantillon à la fois. <p class="jove_conten...

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolement de cellules murines ganglions lymphatiques stromales

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

isolation-of-murine-lymph-node-stromal-cells

Research

JoVE Journal

Immunology and Infection

31.0K vues.  University of Basel and University Hospital Basel. Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells. 

Vidéo: Isolation of Murine Lymph Node Stromal Cells

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Microscopie intravitale du ganglion lymphatique inguinal

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

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Immunologie et infection

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex vivo l&#39;imagerie de cellules T dans murin tranches avec des ganglions lymphatiques et la microscopie confocale Widefield

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

Murine chirurgie superficielle des ganglions lymphatiques

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagerie intravitale du ganglion lymphatique poplité souris

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Myéloïde cellule d&#39;isolement de la peau de souris et Vidange ganglion Après intradermique immunisation avec Live Atténuée<em&gt; Plasmodium</em&gt; sporozoïtes

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Collecte et traitement des ganglions lymphatiques des animaux de grande taille pour l’analyse de l’ARN : préparation pour l’étude de transcriptomique ganglion des grandes espèces animales

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Détermination des sous-ensembles de lymphocytes T régulateurs dans le Thymus murin, pancréatique, drainage des ganglions lymphatiques et la rate à l’aide de cytométrie en flux

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Isolement des sous-ensembles macrophages et des cellules stromales des spécimens myocardiques humains et de souris

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.