Wir bedanken uns bei Sylvie Lesage und alle Labor-Mitglieder für die kritische Durchsicht des Protokolls. Diese Arbeit wurde durch ein Stipendium (MOP-77545) von der kanadischen Institutes of Health Research (CIHR) unterstützt. Nathalie Labrecque wird durch ein Stipendium unterstützt FRSQ Senioren und Mélissa Mathieu erhielt eine NSERC Alexander Graham Bell Kanada Graduate Scholarship.

1. Vorbereitung Vor der Operation Die Mäuse wurden in Übereinstimmung mit den kanadischen Rat am Animal Care-Richtlinien behandelt. <ol><li> Anästhesieren Mäusen durch Injektion von Ketamin / Xylazin (150/300 mg / kg, ip) oder durch Inhalation von Isofluran (2%, 1L Sauerstoff). Sofern vorhanden, sollte Isofluran verwendet, da es für eine bessere Kontrolle der Narkose Zeit und Tiefe ermöglicht werden. Auch wird der Sauerstoff zur gleichen Zeit, die die physiologischen Funktionen des Tieres verabreicht unterstützt. Darüber hinaus ist Isofluran direkt von den Lungen eliminiert und erfordert daher keine Leber oder Niere Stoffwechsel. </li><li> Inject Buprenorphin (0,05 - 0,1 mg / kg, sc) oder ein anderes Analgetikum. </li><li> Anwenden Salbe auf die Mausaugen vor der Operation Augentrockenheit zu vermeiden (wenn Isofluran verwendet wird). </li><li> Stellen Sie sicher, dass die Maus schläft durch Kneifen der Pfote und rollen mit dem Schwanz. Wenn die Maus nicht reagiert, mit der Operation fortzufahren. </li></ol> 2. Die Chirurgie <pclass = &quot;jove_content&quot;&gt; Es ist möglich, 4 von den oberflächlichen Lymphknoten (LN) durch eine Operation zu entfernen: die 2 Leisten-und die 2 brachialis. Die Isolierung der LN ist einfacher in jungen schlanken Mäuse (6-8 Wochen), da das Vorhandensein von Fettgewebe bei älteren Mäusen kann mit LN Isolierung stören. Allerdings wird diese Einschränkung mit wiederholter Übung zu überwinden. Für die Mäuse Komfort, ernten wir ein Maximum von 2 LNs zu einer Zeit, und wenn wir Zeit-Gang-Experimente durchzuführen, ernten wir LNs jeden zweiten Tag. Auch eine Infektion zu verhindern, könnten Antibiotika zur Maus gegeben werden, aber wir tun nicht und Wunden werden heilen just fine. Man muss beachten, dass in Abhängigkeit von der experimentellen Design, die Verwendung eines Antibiotikums beeinflussen könnten das Ergebnis beobachtet (z. B. wenn eine Antwort auf ein Bakterium gemessen wird). Beachten Sie, dass alle Instrumente nach jedem Gebrauch sterilisiert werden und aseptischen Bedingungen werden während der Verfahren beibehalten. <ol><li> Legen Sie die Maus auf die Seite. </li><li> Lokalisieren Sie die Region, wo Sie eine incisio durchführen wirdn in Abhängigkeit von der LN Sie ernten. (Abbildung 1). </li><li> Bewerben Chlorhexidin oder ein anderes Desinfektionsmittel, für diese Region. </li><li> Machen Sie einen kleinen Schnitt mit einer scharfen Schere (ca. 5 mm) entweder auf der Rückseite der Vorderbeine oder oberhalb der Hinterpfote in der Nähe des Bauches (Abbildung 1). </li><li> Entfernen Sie die lose Haare du mit der Zange abgeschnitten haben. Wenn Sie möchten, können Sie die Maus vor dem Schnitt zu rasieren. Allerdings verlängert er das Verfahren und wir finden, dass es nicht notwendig, da, wie hier gezeigt, es zu keiner Verbesserung Heilung oder Abnahme Infektion, die beide sind sehr selten. </li><li> Dehnen Sie den Schnitt mit 2 Zangen, um die LN zu sehen. Der Schnitt könnte erreichen 10 mm. Der LN erscheint grau oder dunkler als die umgebende Fett. </li><li> Drücken Sie den Faszien (die dünne Membran, die den Fett-und Gewebe) auf der Oberseite der LN mit einer Zange und ziehen leicht, ohne das umliegende Gewebe. </li><li> Platzieren der zweiten Zange so weit wie möglich underneath den LN. Mit den ersten Pinzette, brechen die Faszie und entfernen Sie den LN. Wenn Sie erfolgreich die LN haben geerntet, sollte ein Blutfleck an der Stelle, wo die LN zuvor befand erscheinen. Beachten Sie, dass die LN versinkt in isotonischer Lösung. Dieser einfache Test erlaubt es zu prüfen, dass Sie ein LN und nicht das Fettgewebe extrahiert. </li><li> Stellen Sie sicher, dass keine Haare in der Wunde sind. </li><li> Die Inzision durch Kleben die 2 Teile der Haut zusammen (die Innenseite der Haut) und durch das Eintreiben sie mit einem Clip. Wir verwenden einige Michel Clip mit den Anlegezange. Installieren eines oder 2 Clips in Abhängigkeit von der Größe des Einschnitts. Meistens fallen die Clips aus, wenn das Gewebe heilt. </li></ol> 3. Postoperative Pflege <ol><li> Fügen Sie etwas Nassfutter an den Boden des Käfigs, so dass die Mäuse leicht aufnehmen können nach der Operation. </li><li> Über 6 bis 8 Stunden nach der Operation, überprüfen Sie die Maus, um sicherzustellen, dass sie keine Schmerzen. Beachten Sie sorgfältig ihr Verhalten (Haltung, Gang, inInteraktion mit der Aromabestandteile und Aussehen des Fells). Wenn das allgemeine Erscheinungsbild der Mäuse ist schlecht, verwalten eine zweite Dosis von Buprenorphin. Am nächsten Tag sollten die Mäuse ihr normales Verhalten sonst wieder weiter die Verabreichung von Buprenorphin. Die Mäuse sollten nie erreichen die Endpunkte zuvor ermittelten und akzeptiert von Ihrer Animal Care Council. Vielleicht möchten Sie beraten zu lassen und dabei die Hilfe eines Technikers Tierpflege Vivarium. </li></ol> 4. Lymph Node Handhabung <ol><li> Im Labor, um eine einzelne Zelle Suspension zu erhalten, distanzieren die LN nach Ihren üblichen Verfahren. Wir drücken LN gegen gefrostet Dias und routinemäßig erhalten 1-2 x 10 6 Zellen pro LN. Alternativ bereiten wir den LN zur RNA-Extraktion. </li><li> Stain die Zellen wie pro Ihre übliche Protokoll. Sie sollten erhalten erwartet LN Durchflusszytometrie Profile. </li></ol> 5. Repräsentative Ergebnisse Ein Beispiel für Vorwärts-und Seitwärts-Streulicht pCharge wird LNs von einem anästhesierten Maus durch eine Operation oder von einer Maus getötet (2) geerntet gezeigt. Das Profil und die Prozentsätze der Zellen in der Live-Lymphozyten sind die gleichen Gate zeigt, dass LN Operation eine geeignete Technik zum Ernten der Zellen ist. <img alt="1" src="/files/ftp_upload/3444/3444fig1.jpg" /> Abbildung 1. Leistenbruch und brachialis LN Lokalisation in der Maus. A. Die inguinalen LN wird knapp oberhalb der Hinterpfote gelegen, leicht in Richtung Bauch. B. Die brachialis LN befindet sich hinter dem Vorderbein entfernt. <img alt="2" src="/files/ftp_upload/3444/3444fig2.jpg" /> Abbildung 2. Durchflusszytometrie Profil von Lymphozyten nach LN Chirurgie erhalten. Vorwärts-und Seitwärts-Streulicht-Profile werden für LNS aus einer Anesth geerntet gezeigtetized Maus durch eine Operation (A) oder von einer Maus getötet (B). LNs wurden geerntet, dissoziiert und analysiert mit einem Durchflusszytometer. Prozentsätze der Zellen in der Live-Lymphozyten Gate dargestellt.

<ol>
	<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. Immunol. 182, 6195-6206 (2009).</li><li>Rooke, R., Waltzinger, C., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+complementation+of+MHC+class+II+deficiency+by+intrathymic+delivery+of+recombinant+adenoviruses.">Targeted complementation of MHC class II deficiency by intrathymic delivery of recombinant adenoviruses.</a> Immunity. 7, 123-134 (1997).</li><li>Witherden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracycline-controllable+selection+of+CD4(+)+T+cells:+half-life+and+survival+signals+in+the+absence+of+major+histocompatibility+complex+class+II+molecules.">Tetracycline-controllable selection of CD4(+) T cells: half-life and survival signals in the absence of major histocompatibility complex class II molecules.</a> J. Exp. 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. Natl. Acad. Sci. U.S.A. 105, 20440-20445 (2008).</li><li>Obst, R., van Santen, H. M., Mathis, D., Benoist, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+persistence+is+required+throughout+the+expansion+phase+of+a+CD4(+)+T+cell+response.">Antigen persistence is required throughout the expansion phase of a CD4(+) T cell response.</a> J. Exp. Med. 201, 1555-1565 (2005).</li><li>Bassett, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T-cell+expansion+and+maintenance+after+recombinant+adenovirus+immunization+rely+upon+cooperation+between+hematopoietic+and+nonhematopoietic+antigen-presenting+cells.">CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells.</a> Blood. 117, 1146-1155 (2011).</li><li>Lacombe, M. H., Hardy, M. P., 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=IL-7+receptor+expression+levels+do+not+identify+CD8++memory+T+lymphocyte+precursors+following+peptide+immunization.">IL-7 receptor expression levels do not identify CD8+ memory T lymphocyte precursors following peptide immunization.</a> J. 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>

Keine Interessenskonflikte erklärt.

Wir haben ein Protokoll von LN Operation, die zu vielen experimentellen Systemen anwenden können, beschrieben. Obwohl sehr nützlich, um Immunantworten oder Immunzellen Homöostase zu folgen, hat diese Technik einige Einschränkungen. Erstens kann nur vier LNs geerntet werden. Zweitens muss die Immunantwort untersucht werden systemische oder treten in den oberflächlichen Leisten-oder Arm-(Haut-Entleerung) LNS. Schließlich wird die Anzahl der Zellen geerntet von der Qualität des LN Entfernung, eine technische Beschr?...

<table border="1"><tbody><tr><td> Reagenz Namen </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Graefe Feine Muster Premium-Zange </td><td> Harvard Apparatus </td><td> 52-2144 </td><td> Stark gebogen, 10 cm (4 Zoll), 0,8 mm Spitze </td></tr><tr><td> Michel Clip Anwenden und Entfernen von Pinzetten </td><td> Harvard Apparatus </td><td> 52-3779 </td><td> 12,5 cm (5 Zoll) </td></tr><tr><td> Michel Clip 100 </td><td> Harvard Apparatus </td><td> 52-3746 </td><td> 7,5 bis 1,75 mm </td></tr><tr><td> Temgesic (Buprenorphin-Hydrochlorid) </td><td> Schering-Plough </td><td></td><td></td></tr><tr><td> Vetalar (Ketamin Hydrochlorid) </td><td> Boniche Animal Healthcare </td><td></td><td> DIN: 01989529 </td></tr><tr><td> Rompun (Xylazin) </td><td> Bayer </td> &lt;td&gt; </td><td> DIN: 02169592 </td></tr><tr><td> Isofluran </td><td> ABBOTT </td><td></td><td> DIN: 02032384 </td></tr><tr><td> Hypotears Augensalbe </td><td> Novartis Ophtalmics </td><td></td><td> DIN: 02133288 </td></tr><tr><td> Baxedin (Chlorhexidin Gluconat 2% in Isopropylalkohol 70%) </td><td> Omega </td><td> L0000017 </td><td> DIN: 02251477 </td></tr></tbody></table>

murine superficial lymph node surgery

Auf dem Gebiet der Immunologie, um das Fortschreiten einer Immunantwort gegen ein Impfstoff, einer Infektion oder eines Tumors zu verstehen, wird die Antwort oft über die Zeit verfolgt. In ähnlicher Weise erfordert die Untersuchung von Lymphozyten Homöostase Zeitverlauf Experimenten. Durchführung dieser Untersuchungen innerhalb der gleichen Maus ist ideal, um die experimentellen Variabilität sowie die Anzahl der Mäuse zu verringern. Blutentnahmesystem ermöglicht Leistung Zeitverlauf Versuche, aber es gibt nur Informationen über zirkulierenden Lymphozyten und eine begrenzte Anzahl von Zellen 1-4. Da Lymphozyten durch den Körper zirkuliert und die ihren Wohnsitz in den Lymphknoten unterschiedliche Eigenschaften haben, ist es wichtig, beide Standorte zu untersuchen. Die sequentielle Entfernung der Lymphknoten durch eine Operation bietet eine einzigartige Gelegenheit, um eine Immunantwort oder Immunzellen Expansion in der gleichen Maus über die Zeit zu folgen. Darüber hinaus liefert diese Technik zwischen 1-2x10 6 Zellen pro Lymphknoten, die ausreicht, EWrform phänotypischen Charakterisierung und / oder funktionellen Assays. Sequential Lymphknoten Operation oder Lymphadenektomie wurde erfolgreich von uns und anderen 5-11 verwendet. Hier beschreiben wir, wie die brachialen und inguinalen Lymphknoten indem Sie einen kleinen Schnitt in der Haut eines anästhesierten Maus entfernt werden kann. Seit der Operation ist oberflächlich und schnell erledigt, die Maus sehr schnell erholt, heilt gut und nicht die Erfahrung, übermäßige Schmerzen. Alle zwei Tage, ist es möglich, ein oder zwei Lymphknoten Berücksichtigung Zeitverlauf Versuche ernten. Diese Technik eignet sich somit, um die Eigenschaften von Lymphknoten-Lymphozyten Wohnsitz im Laufe der Zeit zu untersuchen. Dieser Ansatz ist geeignet, um verschiedene experimentelle Designs und wir glauben, dass viele Labors würden von der Durchführung sequentieller Lymphknoten-Operationen profitieren können.

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

Murine Superficial Lymph Node Surgery

Um den Verlauf einer Immunantwort im Laufe der Zeit innerhalb der gleichen Maus folgen, können Lymphknoten sequentiell durch eine Operation entfernt werden. Hier beschreiben wir, wie diese Technik durchgeführt werden kann.

Murine Oberflächliche Lymphknoten Chirurgie

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

42.1K Aufrufe.  Maisonneuve-Rosemont Hospital Research Center. Um den Verlauf einer Immunantwort im Laufe der Zeit innerhalb der gleichen Maus folgen, können Lymphknoten sequentiell durch eine Operation entfernt werden. Hier beschreiben wir, wie diese Technik durchgeführt werden kann.

Serie: 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.

Intravitalmikroskopie der Leistenbruch 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 ...

Forschung

Immunologie und Infektion

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-Imaging von T-Zellen in Murine Lymph Node Slices mit Weitfeld und konfokale Mikroskope

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.

Bildgebung des dem Maus-Kniekehlen-Lymphknoten

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.

Generierung von Lymphknoten-Fett-Pad Chimären für die Studie der Lymphknoten 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 ...

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.

Isolierung von murinen Lymphknoten Stromazellen

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.

Myeloischen Zellisolierung von Mäusehaut und ableitenden Lymphknoten Nach intradermale Impfung mit attenuierten Lebend<em&gt; Plasmodium</em&gt; Sporozoiten

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 Vollhauttransplantation

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.

Intrazelluläre Färbung und Durchflusszytometrie zu identifizieren Lymphozytensubpopulationen in Murine Aorta, Niere und Lymphknoten in einem Modell von Hypertonie

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.

Erhebung und Verarbeitung von Lymphknoten von großen Tieren für RNA-Analyse: Vorbereitung auf Lymphknoten Transkriptomischen Studien der großen Tierarten

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.

Ermittlung der regulatorischen T Zelle Teilmengen in murinen Thymus, Pankreas, Entwässerung, Lymphknoten und Milz mit Durchflusszytometrie

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, ...