Nous tenons à remercier Sylvie Lesage et tous les membres du laboratoire pour la lecture critique du protocole. Ce travail a été soutenu par une subvention (MOP-77545) à partir de l&#39;Institut canadien de recherche en santé (IRSC). Nathalie Labrecque est soutenu par une bourse du FRSQ principal et Mélissa Mathieu a reçu une CRSNG Alexander Graham Bell Canada Bourses d&#39;études supérieures.

1. Préparation avant la chirurgie Les souris ont été traitées conformément au Conseil canadien sur les lignes directrices en soins des animaux. <ol><li> Anesthésie souris par injection de kétamine / xylazine (150/300 mg / kg, ip) ou par inhalation d&#39;isoflurane (2%, l&#39;oxygène 1L). Si elle est disponible, l&#39;isoflurane doit être utilisé car il permet un meilleur contrôle de la durée de l&#39;anesthésie et la profondeur. En outre, l&#39;oxygène est administré en même temps qui prend en charge les fonctions physiologiques de l&#39;animal. En outre, l&#39;isoflurane est directement éliminé par les poumons et ne nécessite donc pas d&#39;insuffisance hépatique ou rénale du métabolisme. </li><li> Injecter la buprénorphine (0,05 - 0,1 mg / kg, sc) ou un autre analgésique. </li><li> Appliquer une pommade sur les yeux de souris avant la chirurgie pour éviter la sécheresse oculaire (si l&#39;isoflurane est utilisé). </li><li> Vérifiez que la souris est endormi en pinçant sa patte et rouler sa queue. Si la souris ne réagit pas, procéder à la chirurgie. </li></ol> 2. La chirurgie <pclass = &quot;jove_content&quot;&gt; Il est possible de retirer 4 des ganglions lymphatiques superficiels (LN) par la chirurgie: le inguinaux 2 et le brachial 2. L&#39;isolement de la LN est plus facile chez les jeunes souris maigres (6-8 semaines) depuis la présence de tissu adipeux dans les souris plus âgées peuvent interférer avec l&#39;isolement LN. Toutefois, cette limitation est dépassée à la pratique répétée. Pour le confort des souris, nous récoltons un maximum de 2 ganglions à un moment et quand nous effectuons le temps de cours des expériences, nous récoltons tous les deux jours ganglions. En outre, pour prévenir l&#39;infection, les antibiotiques pourraient être donnés à la souris, mais nous ne faisons pas et les plaies se cicatrisent très bien. Il faut être conscient du fait que selon la conception expérimentale, l&#39;utilisation d&#39;un antibiotique pourrait influencer le résultat observé (par exemple si une réponse à une bactérie est mesurée). Notez que tous les instruments sont stérilisés après chaque utilisation et des conditions d&#39;asepsie sont maintenus tout au long des procédures. <ol><li> Placez la souris sur le côté. </li><li> Localiser la région où vous effectuez une incision, en fonction de la LN vous voulez récolter. (Figure 1). </li><li> Appliquer la chlorhexidine, ou un autre désinfectant, dans cette région. </li><li> Faire une petite incision avec des ciseaux pointus (environ 5 mm), soit sur ​​le dos de la patte avant ou au-dessus de la patte arrière près de l&#39;abdomen (figure 1). </li><li> Retirer les poils que vous avez coupés avec des pinces. Si vous préférez, vous pouvez raser la souris avant de faire l&#39;incision. Cependant, il allonge la procédure et nous constatons qu&#39;il n&#39;est pas nécessaire puisque, comme indiqué ici, il n&#39;améliore pas l&#39;infection de guérison ou de la diminution, qui sont à la fois très rare. </li><li> Étirez l&#39;incision avec une pince à 2 afin de voir le LN. L&#39;incision peut atteindre 10 mm. La LN apparaît grisâtre ou plus foncée que la graisse environnante. </li><li> Pincer le fascia (la membrane mince couvrant la graisse et de tissus) sur le dessus de la LN avec un pince et tirer légèrement sans rompre le tissu environnant. </li><li> Placez la pince deuxième, autant que possible de l&#39;UEDNrneath la LN. Avec la pince premiers, briser le fascia et retirez la LN. Si vous avez réussi à récolter la LN, une tache de sang devrait apparaître à l&#39;endroit où l&#39;LN se trouvait précédemment. Notez que les puits de LN en solution isotonique. Ce test simple permet de valider que vous avez extrait une LN et les tissus non gras. </li><li> Vérifiez qu&#39;aucun poils sont dans la plaie. </li><li> Fermer l&#39;incision par collage des parties de la peau 2 ensemble (l&#39;intérieur de la peau) et en les agrafage avec un clip. Nous utilisons un certain Michel clip avec les pinces applicables. Installer un ou deux pinces selon la taille de l&#39;incision. Le plus souvent, les clips de tomber lorsque le tissu guérit. </li></ol> 3. Soins post-opératoires <ol><li> Ajoutez un peu de nourriture humide au fond de la cage de sorte que les souris peuvent se nourrir facilement après la chirurgie. </li><li> Environ 6 à 8 heures après la chirurgie, vérifiez les souris afin de s&#39;assurer qu&#39;ils ne connaissent pas la douleur. Observez attentivement leur comportement (posture, la démarche, enteraction avec congénère et l&#39;apparence de la fourrure). Si l&#39;apparence générale des souris est pauvre, d&#39;administrer une deuxième dose de buprénorphine. Dès le lendemain, les souris doivent retrouver leur comportement normal sinon poursuivre l&#39;administration de la buprénorphine. Les souris ne doit jamais atteindre les critères d&#39;évaluation préalablement définis et acceptés par votre Conseil de protection des animaux. Vous voudrez peut-être demander des conseils et l&#39;aide de votre technicien vivarium des soins aux animaux. </li></ol> 4. Manipulation des ganglions lymphatiques <ol><li> Dans le laboratoire, pour obtenir une suspension cellulaire unique, dissocier la LN selon la procédure habituelle. Nous appuyer sur LN contre diapositives givrées et obtenir systématiquement 1-2 x 10 6 cellules par LN. Sinon, nous préparons la LN pour l&#39;extraction de l&#39;ARN. </li><li> Colorer les cellules selon le protocole habituel. Vous devriez obtenir attendus profils LN cytométrie en flux. </li></ol> 5. Les résultats représentatifs Un exemple de diffusion vers l&#39;avant et p côtélot étant indiqué pour LNS récoltées à partir d&#39;une souris anesthésiée par la chirurgie ou d&#39;une souris sacrifiées (figure 2). Le profil et les pourcentages de cellules dans la grille en direct des lymphocytes sont les mêmes qui démontre que la chirurgie LN est une technique appropriée pour les cellules de la récolte. <img alt="Figure 1" src="/files/ftp_upload/3444/3444fig1.jpg" /> Figure 1. Inguinale et la localisation LN brachial chez la souris. A. La LN inguinale est situé juste au-dessus de la patte arrière, légèrement vers le ventre. B. Le LN brachiale est situé derrière la patte antérieure. <img alt="Figure 2" src="/files/ftp_upload/3444/3444fig2.jpg" /> Figure 2. Profil cytométrie de flux des lymphocytes obtenus suite à une chirurgie LN. Profils de dispersion avant et latérale sont indiqués pour LNS récoltées à partir d&#39;un Anesthla souris etized par la chirurgie (A) ou d&#39;une souris sacrifiées (B). LN ont été récoltés, dissociés et analysés sur un cytomètre de flux. Les pourcentages de cellules dans la grille en direct des lymphocytes sont présentés.

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	<li>Zehn, D., Lee, S. Y., Bevan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+but+curtailed+T-cell+response+to+very+low-affinity+antigen.">Complete but curtailed T-cell response to very low-affinity antigen.</a> Nature. 458, 211-214 (2009).</li><li>Vezys, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+CD8+T-cell+compartment+grows+in+size+with+immunological+experience.">Memory CD8 T-cell compartment grows in size with immunological experience.</a> Nature. 457, 196-199 (2009).</li><li>Zhou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+persistence+of+memory+CD8(+)+T+cells+depend+on+T+cell+factor+1.">Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1.</a> Immunity. 33, 229-240 (2010).</li><li>Wirth, T. C., Badovinac, V. P., Zhao, L., Dailey, M. O., Harty, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+central+memory+CD8+T+cells+is+independent+of+CD62L-mediated+trafficking+to+lymph+nodes.">Differentiation of central memory CD8 T cells is independent of CD62L-mediated trafficking to lymph nodes.</a> J. 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Med. 191, 355-364 (2000).</li><li>Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+much+TCR+does+a+T+cell+need.">How much TCR does a T cell need.</a> Immunity. 15, 71-82 (2001).</li><li>Leignadier, J., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+density+influences+CD8++memory+T+cell+differentiation.">Epitope density influences CD8+ memory T cell differentiation.</a> PLoS One. 5, e13740 (2010).</li><li>Leignadier, J., Hardy, M. P., Cloutier, M., Rooney, J., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+T-lymphocyte+survival+does+not+require+T-cell+receptor+expression.">Memory T-lymphocyte survival does not require T-cell receptor expression.</a> Proc. 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Immunol. 175, 4400-4407 (2005).</li><li>Leignadier, J., Rooney, J., Daudelin, J. F., Labrecque, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lowering+TCR+expression+on+naive+CD8++T+cells+does+not+affect+memory+T-cell+differentiation.">Lowering TCR expression on naive CD8+ T cells does not affect memory T-cell differentiation.</a> Immunol. Cell. Biol. 89, 322-325 (2011).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Nous avons décrit un protocole de chirurgie LN qui peut s&#39;appliquer à de nombreux systèmes expérimentaux. Bien que très utile pour suivre les réponses immunitaires ou l&#39;homéostasie des cellules immunitaires, cette technique a quelques limitations. Tout d&#39;abord, seulement quatre ganglions peuvent être récoltés. Deuxièmement, la réponse immunitaire étudiée doit être systémique ou se produire dans les inguinaux superficiels ou brachiale (la peau de vidange) LNS. Enfin, le nombre de cellules réc...

<table border="1"><tbody><tr><td> Nom réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Graefe Beaux Forceps haut de gamme de modèle </td><td> Harvard Apparatus </td><td> 52-2144 </td><td> Fortement courbé, 10 cm (4 po), 0,8 mm pointe </td></tr><tr><td> Clips Michel application et le retrait Forceps </td><td> Harvard Apparatus </td><td> 52-3779 </td><td> 12,5 cm (5 po) </td></tr><tr><td> Clips Michel 100 </td><td> Harvard Apparatus </td><td> 52-3746 </td><td> De 7,5 à 1,75 mm </td></tr><tr><td> Temgésic (chlorhydrate de buprénorphine) </td><td> Schering-Plough </td><td></td><td></td></tr><tr><td> Vetalar (chlorhydrate de kétamine) </td><td> Boniche animale Santé </td><td></td><td> DIN: 01989529 </td></tr><tr><td> Rompun (xylazine) </td><td> Bayer </td> &lt;td&gt; </td><td> DIN: 02169592 </td></tr><tr><td> L&#39;isoflurane </td><td> ABBOTT </td><td></td><td> DIN: 02032384 </td></tr><tr><td> Pommade oculaire Hypotears </td><td> Novartis ophtalmiques </td><td></td><td> DIN: 02133288 </td></tr><tr><td> Baxedin (gluconate de Clorhexidine 2% dans l&#39;alcool isopropylique à 70%) </td><td> Oméga </td><td> L0000017 </td><td> DIN: 02251477 </td></tr></tbody></table>

murine superficial lymph node surgery

Dans le domaine de l&#39;immunologie, de comprendre la progression d&#39;une réponse immunitaire contre un vaccin, une infection ou une tumeur, la réponse est souvent suivie au fil du temps. De même, l&#39;étude de l&#39;homéostasie lymphocytaire nécessite des expériences de cours à temps. La réalisation de ces études dans la même souris est idéale pour réduire la variabilité expérimentale ainsi que le nombre de souris utilisées. De prélèvement de sang permet des performances des expériences de cours à temps, mais il ne donne que des informations sur les lymphocytes circulants et fournit un nombre limité de cellules 1-4. Depuis lymphocytes circulants dans le corps et résidant dans les ganglions lymphatiques ont des propriétés différentes, il est important d&#39;examiner ces deux endroits. La suppression séquentielle des ganglions lymphatiques par la chirurgie offre une occasion unique de suivre une réponse immunitaire ou l&#39;expansion des cellules immunitaires chez la souris même au fil du temps. En outre, cette technique donne entre 1-2x10 6 cellules par des ganglions lymphatiques qui est suffisant pour perform caractérisation phénotypique et / ou des tests fonctionnels. Séquentiel chirurgie ganglionnaire ou lymphadénectomie a été utilisé avec succès par nous et les autres 5-11. Ici, nous décrivons comment les ganglions lymphatiques brachial et inguinales peuvent être retirés en faisant une petite incision dans la peau d&#39;une souris anesthésiée. Depuis la chirurgie est superficielle et fait rapidement, la souris récupère très rapidement, guérit bien et ne pas éprouver de la douleur excessive. Tous les deux jours, il est possible de récolter un ou deux ganglions lymphatiques permettant des expériences de cours à temps. Cette technique est donc appropriée pour étudier les caractéristiques des ganglions lymphatiques des lymphocytes résidant au fil du temps. Cette approche est adaptée aux différents modèles expérimentaux et nous pensons que de nombreux laboratoires gagneraient à effectuer séquentielles chirurgies des ganglions lymphatiques.

Watch this Scientific Journal Video about Murine Superficial Lymph Node Surgery at JoVE.com

Murine Superficial Lymph Node Surgery

Pour suivre la progression d&#39;une réponse immunitaire au fil du temps au sein de la même souris, des ganglions lymphatiques peut être enlevé par chirurgie séquentielle. Ici, nous décrivons comment cette technique peut être réalisée.

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 ...

Research

JoVE Journal

Immunology and Infection

42.0K vues.  Maisonneuve-Rosemont Hospital Research Center. Pour suivre la progression d'une réponse immunitaire au fil du temps au sein de la même souris, des ganglions lymphatiques peut être enlevé par chirurgie séquentielle. Ici, nous décrivons comment cette technique peut être réalisée.

Vidéo: Murine Superficial Lymph Node Surgery

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 ...

Recherche

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 ...

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 ...

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.

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 ...

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 ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine Transplantation de peau pleine épaisseur

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

Intracellulaires Coloration et cytométrie de flux pour identifier lymphocytaires sous-ensembles au sein murin Aorte, Reins et les ganglions lymphatiques dans un modèle de l&#39;hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

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, ...