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>
	<li>Mueller, S. N., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphoid+stroma+in+the+initiation+and+control+of+immune+responses.">Lymphoid stroma in the initiation and control of immune responses.</a> Immunological reviews. 224, 284-294 (2008).</li><li>Roozendaal, R., Mebius, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+Cell+-+Immune+Cell+Interactions.">Stromal Cell - Immune Cell Interactions.</a> Annual Review of Immunology. 29 (1), 23-43 (2011).</li><li>Turley, S. J., Fletcher, A. L., Elpek, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stromal+and+haematopoietic+antigen-presenting+cells+that+reside+in+secondary+lymphoid+organs.">The stromal and haematopoietic antigen-presenting cells that reside in secondary lymphoid organs.</a> Nature Reviews Immunology. 10 (12), (2010).</li><li>Baj&#233;noff, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell+networks+regulate+lymphocyte+entry,+migration,+and+territoriality+in+lymph+nodes.">Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes.</a> Immunity. 25 (6), 989-1001 (2006).</li><li>Katakai, T., Hara, T., Sugai, M., Gonda, H., Shimizu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+fibroblastic+reticular+cells+construct+the+stromal+reticulum+via+contact+with+lymphocytes.">Lymph node fibroblastic reticular cells construct the stromal reticulum via contact with lymphocytes.</a> The Journal of experimental medicine. 200 (6), 783-795 (2004).</li><li>Link, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblastic+reticular+cells+in+lymph+nodes+regulate+the+homeostasis+of+naive+T+cells.">Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells.</a> Nature Immunology. 8 (11), 1255-1265 (2007).</li><li>Malhotra, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+stroma+from+inflamed+and+resting+lymph+nodes+defines+immunological+hallmarks.">Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.</a> Nature Immunology. 13 (5), 499-510 (2012).</li><li>Gretz, J. E., Norbury, C. C., Anderson, A. O., Proudfoot, A. E., Shaw, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph-borne+chemokines+and+other+low+molecular+weight+molecules+reach+high+endothelial+venules+via+specialized+conduits+while+a+functional+barrier+limits+access+to+the+lymphocyte+microenvironments+in+lymph+node+cortex.">Lymph-borne chemokines and other low molecular weight molecules reach high endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex.</a> The Journal of experimental medicine. 192 (10), 1425-1440 (2000).</li><li>Sixt, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conduit+system+transports+soluble+antigens+from+the+afferent+lymph+to+resident+dendritic+cells+in+the+T+cell+area+of+the+lymph+node.">The conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the T cell area of the lymph node.</a> Immunity. 22 (1), 19-29 (2005).</li><li>MartIn-Fontecha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dendritic+cell+migration+to+the+draining+lymph+node:+impact+on+T+lymphocyte+traffic+and+priming.">Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming.</a> The Journal of experimental medicine. 198 (4), 615-621 (2003).</li><li>Luther, S. A., Tang, H. L., Hyman, P. L., Farr, A. G., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexpression+of+the+chemokines+ELC+and+SLC+by+T+zone+stromal+cells+and+deletion+of+the+ELC+gene+in+the+plt/plt+mouse.">Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (23), 12694-12699 (2000).</li><li>Braun, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Afferent+lymph-derived+T+cells+and+DCs+use+different+chemokine+receptor+CCR7-dependent+routes+for+entry+into+the+lymph+node+and+intranodal+migration.">Afferent lymph-derived T cells and DCs use different chemokine receptor CCR7-dependent routes for entry into the lymph node and intranodal migration.</a> Nature Immunology. 12 (9), 879-887 (2011).</li><li>Fletcher, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+isolation+of+lymph+node+stromal+cells+reveals+site-dependent+differences+in+fibroblastic+reticular+cells.">Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.</a> Frontiers in immunology. 2, 35 (2011).</li><li>Lukacs-Kornek, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+release+of+nitric+oxide+by+nonhematopoietic+stroma+controls+expansion+of+the+activated+T+cell+pool+in+lymph+nodes.">Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes.</a> Nature Immunology. 12 (11), 1096-1104 (2011).</li><li>Siegert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblastic+reticular+cells+from+lymph+nodes+attenuate+T+cell+expansion+by+producing+nitric+oxide.">Fibroblastic reticular cells from lymph nodes attenuate T cell expansion by producing nitric oxide.</a> PLoS ONE. 6 (11), e27618 (2011).</li><li>Khan, O., Headley, M., Gerard, A., Wei, W., Liu, L., Krummel, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cell+priming+by+lymphoid+stroma.">Regulation of T cell priming by lymphoid stroma.</a> PLoS ONE. 6 (11), e26138 (2011).</li><li>Svensson, M., Maroof, A., Ato, M., Kaye, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+direct+local+differentiation+of+regulatory+dendritic+cells.">Stromal cells direct local differentiation of regulatory dendritic cells.</a> Immunity. 21 (6), 805-816 (2004).</li><li>Onder, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell-specific+lymphotoxin-&#946;+receptor+signaling+is+critical+for+lymph+node+and+high+endothelial+venule+formation.">Endothelial cell-specific lymphotoxin-&#946; receptor signaling is critical for lymph node and high endothelial venule formation.</a> The Journal of experimental medicine. 210 (3), 465-473 (2013).</li><li>Fletcher, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+fibroblastic+reticular+cells+directly+present+peripheral+tissue+antigen+under+steady-state+and+inflammatory+conditions.">Lymph node fibroblastic reticular cells directly present peripheral tissue antigen under steady-state and inflammatory conditions.</a> The Journal of experimental medicine. 207 (4), 689-697 (2010).</li><li>Fletcher, A. L., Malhotra, D., Turley, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+stroma+broaden+the+peripheral+tolerance+paradigm.">Lymph node stroma broaden the peripheral tolerance paradigm.</a> Trends in Immunology. 32 (1), 12-18 (2011).</li><li>Cohen, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node-resident+lymphatic+endothelial+cells+mediate+peripheral+tolerance+via+Aire-independent+direct+antigen+presentation.">Lymph node-resident lymphatic endothelial cells mediate peripheral tolerance via Aire-independent direct antigen presentation.</a> Journal of Experimental Medicine. 207 (4), 681-688 (2010).</li><li>Lee, J. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+antigen+display+by+lymph+node+stroma+promotes+T+cell+tolerance+to+intestinal+self.">Peripheral antigen display by lymph node stroma promotes T cell tolerance to intestinal self.</a> Nat Immunol. 8, 181-190 (2006).</li><li>Nichols, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletional+self-tolerance+to+a+melanocyte/melanoma+antigen+derived+from+tyrosinase+is+mediated+by+a+radio-resistant+cell+in+peripheral+and+mesenteric+lymph+nodes.">Deletional self-tolerance to a melanocyte/melanoma antigen derived from tyrosinase is mediated by a radio-resistant cell in peripheral and mesenteric lymph nodes.</a> J Immunol. 179, 993-1003 (2007).</li><li>Gardner, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletional+tolerance+mediated+by+extrathymic+Aire-expressing+cells.">Deletional tolerance mediated by extrathymic Aire-expressing cells.</a> Science. 321, 843-847 (2008).</li><li>Gardner, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrathymic+Aire-expressing+cells+are+a+distinct+bone+marrow-derived+population+that+induce+functional+inactivation+of+CD4++T+cells.">Extrathymic Aire-expressing cells are a distinct bone marrow-derived population that induce functional inactivation of CD4+ T cells.</a> Immunity. 39, 560-572 (2013).</li><li>Magnusson, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+presentation+of+antigen+by+lymph+node+stromal+cells+protects+against+CD8+T-cell-mediated+intestinal+autoimmunity.">Direct presentation of antigen by lymph node stromal cells protects against CD8 T-cell-mediated intestinal autoimmunity.</a> Gastroenterology. 134, 1028-1037 (2008).</li><li>Tewalt, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+endothelial+cells+induce+tolerance+via+PD-L1+and+lack+of+costimulation+leading+to+high-level+PD-1+expression+on+CD8+T+cells.">Lymphatic endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to high-level PD-1 expression on CD8 T cells.</a> Blood. 120 (24), 4772-4782 (2012).</li></ol>

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

isolation of murine lymph node stromal cells

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

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

Research

JoVE Journal

Immunology and Infection

31.0K 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

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 ...

Murine Superficial Lymph Node Surgery

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

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

Murine 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 ...

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 ...

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 ...

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, ...

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

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

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

Isolierung von Makrophagen-Teilmengen und Stromalzellen von Menschlichen und Maus-Myokardproben

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

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

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

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