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.

In diesem Video-Veröffentlichung und Protokoll wurden alle Tierverfahren in Übereinstimmung mit der von der kantonalen Behörde Basel-Stadt, Schweiz zugelassen Tier-Protokoll durchgeführt. 1. Lymphknoten Vorbereitung und Verdauung <ol><li> Pre-Wärme-Wasser in einem Becher auf 37 ° C auf einem Magnetrührer mit Heizplatte. </li><li> Bereiten Basis Medium wie folgt: DMEM-Medium (ohne Pyruvat) mit 2% FCS, 1,2 mM CaCl 2 und Pen / Strep (100 Einheiten Penicillin, 100 ug Streptomycin). </li><li> Sterilisieren alle Dissektionsinstrumente vor dem Gebrauch. </li><li> Einschläfern Lymphknoten Spendermäusen pro CO 2 Ersticken und aseptisch sezieren die Lymphknoten. Sezieren nicht die umgebende Fett. HINWEIS: Dieses Protokoll wird für periphere Haut-Lymphknoten (Leisten, brachial, Achsel) optimiert. </li><li> Platzieren Lymphknoten in einer sterilen Petrischale mit 2 ml eiskaltem basischen Medium. </li><li> Stören den Lymphknoten capsule mit zwei 25-G-Nadeln von 1-ml-Spritze befestigt. </li><li> Übertragen des unterbrochenen Lymphknotengewebe in einem 5 ml-Polypropylen-Rundboden-Röhrchen mit 750 ul basischen Medium mit 1 mg / ml Collagenase IV und 40 ug / ml DNAse I ergänzt </li><li> Fügen Sie einen sterilen Magnetrührer in jeder Röhre. </li><li> Das Röhrchen in das Becherglas mit 37 ° C vorgewärmt Wasser und rühren die Rohre mit einer langsamen Geschwindigkeit (1 Runde / sec) für 30 min. </li><li> Entfernen Sie den Schlauch vom Magnetrührer mit Heizplatte und lassen Lymphknotenfragmente zu begleichen. </li><li> Den Überstand in &quot;nicht-Stromazellen&quot; angereichert vorsichtig entfernen. HINWEIS: Wenn die Analyse der T, B, dendritischen Zellen und CD45 - gp38 - CD31 - vorgesehen ist, speichern Sie die &quot;nicht-Stromazellen&quot; Fraktion. </li><li> Wasch übrigen Lymphknotengewebe einmal mit 750 ul Basismedium. Hinweis: Dieser Schritt nur erforderlich, wenn die Arbeit mit immunkompetenten Mäusen. </li><li> Lassen Lymphknotenfragmente zu begleichen. </li><li> Entfernen Sie dieNicht-Stromazellen-Schwimmfraktion. </li><li> In den Lymphknoten-Fragmente 750 ul basischen Medium mit 3,5 mg ergänzt / ml Collagenase D und 40 ug / ml DNase I </li><li> Ort Rohr zurück in den Becher mit dem 37 ° C vorgewärmt Wasser. </li><li> Verdauen Lymphknotengewebe für 5 min, während langsam umrühren. </li><li> Disaggregierten Lymphknotengewebefragmente durch Pipettieren und Mischen 700 ul für 10 Zyklen bei maximaler Geschwindigkeit mit Hilfe eines automatisierten Mehrkanalpipette. HINWEIS: Dies stört Lymphknotengewebe, um die Verdauung zu verbessern. </li><li> Das Röhrchen zurück in das Becherglas mit 37 ° C vorgewärmte Wasser. </li><li> Digest Lymphknotengewebefragmente für weitere 10 min unter langsamem Rühren. </li><li> Disaggregate Lymphknotengewebefragmente durch Pipettieren und Mischen für 99 Zyklen bei maximaler Geschwindigkeit mit Hilfe eines automatisierten Mehrkanalpipette. </li><li> Fügen 7,5 ul 0,5 M EDTA zur Aufrechterhaltung Einzelzellsuspension zu gewährleisten. </li><li> Disaggregierten Lymphknotengewebefragmente von RohrArmatur und Mischen für 99 Zyklen bei maximaler Geschwindigkeit unter Verwendung eines automatisierten Mehrkanalpipette. </li><li> Fügen Sie 750 ul Basismedium und übergeben Zellen durch eine 70 um Nylon-Mesh. </li><li> Zentrifuge Zellsuspension 5 min bei 1.500 × g, 4 ° C beträgt. </li><li> Stromazellen können nun für die weitere Analyse verwendet werden. </li></ol> 2. Die Färbung der Lymphknoten Stromazellen <ol><li> Verwenden Sie eine Kombination von Anti-CD45, Anti-podoplanin (gp38), Anti-CD31-Antikörper gegen TRC, LEC, BEC und DN Zellen erfolgreich zu erkennen. </li><li> Inkubieren der verdauten Lymphknotengewebe mit Live / Dead-Tracker, anti-CD45-FITC (1: 200), anti-gp38-PE (1: 200), Anti-CD31-APC (1: 200) in 100 ul HBSS, enthaltend 2 % FCS für mindestens 20 min, 4 ° C im Dunkeln. </li><li> Waschen Sie die Zellen, indem 500 l HBSS, enthaltend 2% FCS </li><li> Zentrifuge Zellsuspension 3 Minuten bei 1500 × g, 4 ° C beträgt. </li><li> Resuspendieren in 100 ul HBSS, enthaltend 2% FCS. </li><li> Führen Sie die gefärbten Zellen in einem Durchflusszytometer equipped mit folgenden Optik: Anregungsquelle mit bis zu drei Laser: blau (488 nm, luftgekühlt, 20 mW Festkörper), Rot (633 nm, 17 mW HeNe) und Violett (405 nm, 30 mW Festkörper) . </li><li> Tor CD45 - Zellen, die blutbildenden Zellen aus. </li><li> Tor Slips (FSC-W) und lebenden Zellen (LIVE / DEAD-Tracker), um Dubletten und tote Zellen auszuschließen. </li><li> , LEC (gp38 + CD31 +), BEC (gp38 - CD31 +) und DN (gp38 - CD31 - Zellen) (siehe Abbildung 1) - Inhalts gp38 gegen CD31 zu TRC (gp38 + CD31) zu visualisieren. </li></ol>

<ol>
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The authors declare they have no competing financial interests.

Die Studie von Lymphknoten Stromazellen vor kurzem wurde ein Forschungsschwerpunkt auf die Entwicklung von zwei Protokolle veröffentlicht Verdauung 6,13. Beide Protokolle angemessen sind, um einzelne Lymphknoten Stromazellen zu erhalten, unterscheiden sich jedoch in der Verwendung von Verdauungsenzymen und dem Zeitpunkt der Verdauung. Da Stromazellen und ihre Oberflächenmarker empfindlich enzymatischen Verdau und mechanischer Belastung sind, wird ein optimiertes Protokoll erforderlich. <p class="jove_co...

Lymphknoten sind spezialisierte Fächer, wo adaptiven Immunantwort gegen fremde und Selbst-Antigene initiiert und koordiniert. Das hier vorgestellte Verfahren beschreibt eine kurze enzymatische Verdauung kombiniert mit automatisierten mechanischen Pipettieren Lymphknoten Einzelzellsuspension zu erhalten und Zugang zu lebensfähigen Lymphknoten Stromazellen, die die Oberflächenexpression von mehreren Molekülen zu erhalten. Lymphknoten Stromazellen bilden das Gerüst des Lymphknotens und erfüllen drei wichtige Funktionen: sie filtern ersten Körperflüssigkeiten, Antigene, Krankheitserreger und ihre Erregermuster (PAMPs) sowie Zytokine und Gefahr assoziierte molekulare Muster zu probieren (gedämpft) vorhanden im Körper. Zweitens, sie ziehen und weisen Antigen-präsentierenden Zellen (APC) und Lymphozyten zu interagieren und zu initiieren adaptive Immunantworten; und die dritte, bieten sie eine strukturelle Umgebung für die Homöostase und Differenzierung von Lymphozyten 1-3. Während der Entzündung Lymphknoten Stromazellen produzieren Wachstumsfaktoren, Zytokine und Chemokine, die Anpassung an Schwellungen damit die Organisation der Interaktion zwischen dendritischen Zellen (DCs), T-und B-Zellen. Die Instrumentation von Immunantworten ist aufgrund der komplexen Strukturarchitektur von unterschiedlichen stromalen Zellpopulationen gebildet möglich. Lymphknoten Stromazellen CD45-negativen Zellen und kann durch die Expression von CD31 und gp38 in Fibroblasten und Endothelzellen 1 -6 unterscheiden. Gp38 + CD31 - T-Zone definiert Retikulumzellen (TRC, die auch als FRC bekannt: fibroblastischen Retikulumzellen), gp38 + CD31 + definiert lymphatischen Endothelzellen (LEC), gp38 - CD31 + definiert Blut Endothelzellen (BEC). Weitere Charakterisierung von Subpopulationen zeigte die Existenz von anderen Lymphknoten Stromazellen. In der Tat wurde eine kleine pericyte artigen Zellpopulation innerhalb der gekennzeichnetgp38 - CD31 - Population 7. Daher ist die Anpassung des Isolationsverfahren zur Identifizierung und Charakterisierung der funktionellen Eigenschaften von verschiedenen Lymphknoten Stromazellen vorteilhaft. Vor der Entwicklung von Lymphknoten Stromazellen Verdauung Protokolle die Untersuchung von Lymphknoten Stromazellen wurde in situ Beobachtungen mit Gewebeabschnitt und Mikroskopie beschränkt. Dennoch zeigte strukturelle und funktionelle Untersuchungen wichtige Eigenschaften von Lymphknoten Stromazellen. Lymphknoten Stromazellen mit podoplanin, Kollagen und extrazellulärer Matrix (ECM) Proteinen verbunden, um eine komplexe dreidimensionale Struktur 3 genannte Leitungssystem, die Lymphe und assoziierten-niedermolekulare Proteine ​​auf die hohe endotheliale Transporte vom subkapsuläre Sinus des Lymphknotens bilden Venolen in der T-Zellen-Zone 8. DCs sind in engem Kontakt mit Stroma-Zellen und in das rohrförmige con vorstehenden beachtenDuit Struktur zu Probenflüssigkeit und erkennen Antigene 8. Die Wechselwirkung von Lymphknoten Stromazellen (TRCs und LECs) mit DCs ist durch die Freisetzung und Präsentation von Chemokinen CCL21 und CCL19 9,10 vermittelt. CCL19 und CCL21 werden von der CCR7 Rezeptor erleichtert DCs und T-Zellen zu den Lymphknoten T-Zell-Zone 4,11 migrieren anerkannt. Trotz Verwendung ähnlicher Chemokine DCs und T-Zellen haben unterschiedliche Wanderungs in die Lymphknoten 12. Später mittels enzymatischer Verdauung der Lymphknoten und Isolierung von reinem Lymphknoten Stromazellen, funktionelle Studien über die Rolle der verschiedenen Lymphknoten Stromazellen und ihre Fähigkeit, mit DCs und T / B-Zellen interagieren 6,13 durchgeführt. Zuerst wird das Übersprechen zwischen IFN-γ produzierenden T-Effektorzellen und Lymphknoten Stromazellen induziert die Produktion der gezeigten T-Zell-Antworten und die Proliferation in den sekundären lymphoiden Organen 14-16 dämpfen Metaboliten Stickoxid. Zweiten, Lymphe node Stromazellen wurde berichtet, dass die Differenzierung der DC regulatorischen Untergruppen über die Produktion von IL-10-17 zu unterstützen und naiven T-Zell-Homöostase durch die Produktion von IL-7 6,18 modulieren. Drittens TLR-Expression in Lymphknoten stromalen Zellen legt nahe, dass Stromazellen sind anfällig für eine Infektion oder selbst Moleküle während Gewebeverletzung freigesetzt abgeleiteten Signals. Tatsächlich ist die Behandlung von Lymphknoten Stromazellen mit dem Liganden von TLR3 Poly (I: C) induziert eine geringe Hochregulation von Haupthistokompatibilitätskomplex-Klasse-I-Expression und die Hochregulation von Co-inhibitorische Molekül PD-L1, jedoch nicht von kostimulatorischen Molekülen, was zu dramatische Veränderungen im peripheren Gewebe-Antigene Ausdruck 19. Mehrere Gruppen haben gezeigt, Lymphknoten Stromazellen exprimieren peripheren Gewebe-Antigene und induzieren Toleranz von selbstreaktiven T-Zellen 19,21-27. Daher, das Verständnis der Wechselwirkungen zwischen Lymphknoten Stromazellen und die andere Zugvögel und Wiedersident Lymphknotenzellen werden dabei helfen, neue Zielmoleküle zur Aktivierung oder Unterdrückung von Immunreaktionen während der Entzündung ermöglichen zu finden. Daher wird die Umsetzung der veröffentlichten enzymatische Trennung des Lymphknotens erforderlich. Zuvor veröffentlichten Protokolle verwenden verschiedene Kombinationen von Kollagenase-basierte enzymatische Verdauung mit geringer mechanischer Belastung 6,19,20. Allerdings könnte lange Inkubationen mit Verdauungsenzymen oder andere Kombination von verschiedenen Enzym die Verdauung benötigt, um den Aktivierungsstatus zu analysieren und neue Lymphknoten Stromazellen identifizieren Oberflächenmoleküle abzubauen. Abhängig von der Art der stromalen Zellanalyse, kann die Link-Protocol-Protokoll oder Fletcher besser geeignet sein. Bei dem beschriebenen Verfahren wird eine etwas kürzere enzymatischen Verdau mit automatisierten mechanischen Disaggregation kombiniert, um Oberflächenmarker Abbau von lebensfähigen Lymphknoten Stromazellen minimieren. Diese Vorgehensweise ermöglicht hoch reproduzierbare Isolation undUnterscheidung von Lymphknoten Stroma-Zellpopulationen mit geringer Variabilität und mehr als 95% Lebensfähigkeit. Die frisch isolierten Lymphknoten Stromazellen können direkt für Oberflächenmarker-Expression, Proteinanalyse und Transkriptionsstudien sowie Errichtung Stromazellen Leitungen verwendet, um funktionelle Assays in vitro durchzuführen.

<table><tbody><tr><td>DMEM</td><td>LifeTechnologies-Gibco</td><td>41965-039</td><td></td></tr><tr><td>FCS</td><td></td><td></td><td>Final concentration 2%</td></tr><tr><td>CaCl2</td><td>Sigma</td><td>499609</td><td>Final concentration 1.2 mM</td></tr><tr><td>Collagenase IV</td><td>Worthington Biochemical Corporation</td><td></td><td>&gt;160 units per mg dry weight, use at final concentration of 1 mg/ml</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088882001</td><td>use at 3.5 mg/ml</td></tr><tr><td>DNAse I</td><td>Roche&amp;nbsp;</td><td>11284932001</td><td>use at 40 &amp;micro;g/ml</td></tr><tr><td>Stiring magnets</td><td>FAUST</td><td></td><td>5 mm long-2 mm &amp;oslash;</td></tr><tr><td>Polystyrene round bottom tubes 5 ml</td><td>Falcon-BD Bioscience</td><td></td><td></td></tr><tr><td>Magnetic stirrer with heating function</td><td>IKA-RCT-standard</td><td>9720250</td><td></td></tr><tr><td>Petri dishes 100 mm, sterile</td><td>TPP</td><td>6223201</td><td></td></tr><tr><td>25 G needles</td><td>Terumo</td><td></td><td></td></tr><tr><td>anti mouse CD45 Ab</td><td>Biolegend</td><td></td><td>Clone 30-F11</td></tr><tr><td>anti mouse CD11c Ab</td><td>Biolegend</td><td></td><td>Clone N418</td></tr><tr><td>anti mouse Podoplanin Ab</td><td>Biolegend</td><td></td><td>Clone 8.1.1</td></tr><tr><td>anti mouse CD31 Ab</td><td>Biolegend</td><td></td><td>Clone MEC13.3</td></tr><tr><td>&lt;br/&gt; Eppendorf Xplorer plus, Multichannel</td><td>Eppendorf</td><td>4861 000.821/830</td><td>1.250 &amp;micro;l max. volume</td></tr><tr><td>anti mouse CD140a</td><td>Biolegend</td><td></td><td>Clone APA5</td></tr><tr><td>anti mouse CD80</td><td>Biolegend</td><td></td><td>Clone 16-10A1</td></tr><tr><td>anti mouse CD40</td><td>Biolegend</td><td></td><td>Clone 1C10</td></tr><tr><td>anti mouse I-Ab</td><td>Biolegend</td><td></td><td>Clone AF6-120.1</td></tr><tr><td>anti mouse CD274 (PD-L1)</td><td>Biolegend</td><td></td><td>Clone 10F.9G2</td></tr><tr><td>LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td><td>Invitrogen</td><td>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.

Das vorliegende Protokoll ist ein von Link-et al., 2007 6 mit einer kürzeren Kochzeit (45 min maximal) durch mechanische Disaggregation mit einem automatisierten Mehrkanalpipette veröffentlicht modifizierten Verdauung Protokoll. Darüber hinaus ist das Verfahren eher standardisierte minimiert Abbau von Oberflächenmarkern auf verschiedenen Lymphknoten Stromazellen und ermöglicht die Behandlung von mehr als einer Probe gleichzeitig. Kollagenase IV und Collagenase D Link...

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.

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

31.1K Aufrufe.  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. 

Serie: Isolation of Murine Lymph Node Stromal Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolierung von CD4 + T-Zellen aus Maus-Lymphknoten mit Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Forschung

Biologie

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Isolierung von Lymphozyten aus Maus Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Immunologie und Infektion

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolierung und Differenzierung von Th17 Naive CD4-T-Lymphozyten

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolierung, Identifizierung und Reinigung von Murine Thymusepithelzellen

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolierung und Durchflusszytometrie Charakterisierung von murinen Dünndarms Lymphozyten

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolation und Aktivierung von murinen Lymphozyten

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolieren von Lymphozyten aus der Maus kleinen intestinalen Immunsystems

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Optimierung der Isolierung von Stromazellen aus Thymus und Knochenmark

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Autor im Rampenlicht: Weiterentwicklung der hämatopoetischen Forschung mit Stromazellisolierung für die Einzelzellsequenzierung

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Isolierung von Stromazellen aus hämatopoetischen Organen

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Isolierung von murinen Lymphknoten Stromazellen

Zusammenfassung

Weitere Videos entdecken

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Isolierung von CD4 + T-Zellen aus Maus-Lymphknoten mit Miltenyi MACS Purification

Isolierung von Lymphozyten aus Maus Genital Tract Mucosa

Isolierung und Differenzierung von Th17 Naive CD4-T-Lymphozyten

Generierung von Lymphknoten-Fett-Pad Chimären für die Studie der Lymphknoten Stromal Cell Origin

Isolierung, Identifizierung und Reinigung von Murine Thymusepithelzellen

Isolierung und Durchflusszytometrie Charakterisierung von murinen Dünndarms Lymphozyten

Isolation und Aktivierung von murinen Lymphozyten

Isolieren von Lymphozyten aus der Maus kleinen intestinalen Immunsystems

Isolierung von Stromazellen aus hämatopoetischen Organen

Isolierung von Stromazellen aus hämatopoetischen Organen