Name: examination of thymic positive and negative selection by flow cytometry
Uploaded: 2012-10-08T13:00:00
Description: Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Die Autoren bedanken sich bei Bing Zhang für seine technische Unterstützung danken. Diese Arbeit wurde von der kanadischen Institute for Health Research (MOP-86595) finanziert. TAB ist eine CIHR New Investigator und AHFMR Scholar. Doctoral und einem AIHS Vollzeit Studentship - QH wird durch eine CIHR Canada Graduate Scholarship unterstützt. SAN wird von einer Königin Elizabeth II Graduate Scholarship unterstützt. Doctoral - AYWS wird durch ein NSERC Postgraduate-Stipendium unterstützt.

Siehe Abbildung 1 für eine allgemeine Regelung des experimentellen Protokolls Abbildung. Ein. Präparation <ol><li> Ort sterile Stahlgitter Bildschirm in 60 x 15 mm Petrischale. Eine Einheit wird pro Gewebeprobe nötig. </li><li> 5 ml balanced salt Hank-Lösung (HBSS) zu jedem Gericht. Halten Sie Speisen auf dem Eis. </li><li> Einschläfern Mäuse mit CO 2. </li><li> Sichere Maus Dissektion Oberfläche Bauchseite nach oben. Spray-Maus mit 70% E...

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	<li>Liston, A., Lesage, S., Wilson, J., Peltonen, L., Goodnow, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aire+regulates+negative+selection+of+organ-specific+T+cells.">Aire regulates negative selection of organ-specific T cells.</a> Nat. Immunol. 4, 350-354 (2003).</li><li>Liston, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+dosage--limiting+role+of+Aire+in+thymic+expression,+clonal+deletion,+and+organ-specific+autoimmunity.">Gene dosage--limiting role of Aire in thymic expression, clonal deletion, and organ-specific autoimmunity.</a> J. Exp. Med. 200, 1015-1026 (2004).</li><li>Hogquist, K. A., Baldwin, T. A., Jameson, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytes+between+the+beta-selection+and+positive+selection+checkpoints+are+nonresponsive+to+IL-7+as+assessed+by+STAT-5+phosphorylation.">Thymocytes between the beta-selection and positive selection checkpoints are nonresponsive to IL-7 as assessed by STAT-5 phosphorylation.</a> J. Immunol. 172, 4235-4244 (2004).</li><li>Ueno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7+signals+are+essential+for+cortex-medulla+migration+of+developing+thymocytes.">CCR7 signals are essential for cortex-medulla migration of developing thymocytes.</a> J. Exp. 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Immunol. 183, 7761-7767 (2009).</li><li>Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M., von Boehmer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+in+T-cell-receptor+transgenic+mice+involves+deletion+of+nonmature+CD4+8++thymocytes.">Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes.</a> Nature. 333, 742-746 (1988).</li><li>McCaughtry, T. M., Baldwin, T. A., Wilken, M. S., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+deletion+of+thymocytes+can+occur+in+the+cortex+with+no+involvement+of+the+medulla.">Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla.</a> J. Exp. Med. 205, 2575-2584 (2008).</li><li>Derbinski, J., Schulte, A., Kyewski, B., Klein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promiscuous+gene+expression+in+medullary+thymic+epithelial+cells+mirrors+the+peripheral+self.">Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self.</a> Nat. Immunol. 2, 1032-1039 (2001).</li><li>Anderson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projection+of+an+immunological+self+shadow+within+the+thymus+by+the+aire+protein.">Projection of an immunological self shadow within the thymus by the aire protein.</a> Science. 298, 1395-1401 (2002).</li><li>Kurts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+class+I-restricted+exogenous+presentation+of+self+antigens+in+vivo.">Constitutive class I-restricted exogenous presentation of self antigens in vivo.</a> J. Exp. Med. 184, 923-930 (1996).</li><li>Nitta, T., Nitta, S., Lei, Y., Lipp, M., Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR7-mediated+migration+of+developing+thymocytes+to+the+medulla+is+essential+for+negative+selection+to+tissue-restricted+antigens.">CCR7-mediated migration of developing thymocytes to the medulla is essential for negative selection to tissue-restricted antigens.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 17129-17133 (2009).</li><li>Bouneaud, C., Kourilsky, P., Bousso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+negative+selection+on+the+T+cell+repertoire+reactive+to+a+self-peptide:+a+large+fraction+of+T+cell+clones+escapes+clonal+deletion.">Impact of negative selection on the T cell repertoire reactive to a self-peptide: a large fraction of T cell clones escapes clonal deletion.</a> Immunity. 13, 829-840 (2000).</li><li>Gallegos, A. M., 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=Central+tolerance+to+tissue-specific+antigens+mediated+by+direct+and+indirect+antigen+presentation.">Central tolerance to tissue-specific antigens mediated by direct and indirect antigen presentation.</a> J. Exp. Med. 200, 1039-1049 (2004).</li><li>Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+CD4(+)+T+cell+frequency+varies+for+different+epitopes+and+predicts+repertoire+diversity+and+response+magnitude.">Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.</a> Immunity. 27, 203-213 (2007).</li><li>Bouillet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BH3-only+Bcl-2+family+member+Bim+is+required+for+apoptosis+of+autoreactive+thymocytes.">BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes.</a> Nature. 415, 922-926 (2002).</li><li>Suen, A. Y., Baldwin, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proapoptotic+protein+Bim+is+differentially+required+during+thymic+clonal+deletion+to+ubiquitous+versus+tissue-restricted+antigens.">Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens.</a> Proceedings of the National Academy of Sciences of the United States of America. , (2012).</li><li>Calnan, B. J., Szychowski, S., Chan, F. 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Die hier vorgestellten Protokoll kann verwendet werden, um positive und negative Selektion in nicht-transgenen und TCR TCR-transgenen Mäusen zu untersuchen. Dieses Protokoll beschreibt die Färbung von Oberflächen-Antigenen. Für die weitere Analyse der molekularen Mechanismen, ist es oft notwendig, intrazelluläre Färbung führen. Wir verwenden die BD Biosciences Cytofix / Cytoperm Kit für die meisten intrazellulären Proteinen und der BD Biosciences Foxp3 Staining Kit für Transkriptionsfaktoren. Wir in der Regel ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> HyClone Hanks balanced salt solution </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Drahtgewebeelementen </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Petrischalen (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877221 </td><td></td></tr><tr><td> Spritzen (3 ml) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> Konischen Röhren (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Mikroskop </td><td> Zeiss - Primo Star </td><td> 415.500-00XX-000 </td><td></td></tr><tr><td> Hemocytometer </td><td> HausserWissenschaftlich </td><td> 3110 </td><td></td></tr><tr><td> 96-Well-Platte </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Mehrkanalpipette </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> Fötales Kälberserum </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Phosphat-gepufferter Kochsalzlösung </td><td> Fisher Scientific </td><td> SH3025802 </td><td></td></tr><tr><td> Natriumazid </td><td> IT Baker Chemical Co. </td><td> V015-05 </td><td></td></tr><tr><td> FcR Blockierungsreagenz </td><td></td><td></td><td> Clone 2.4G2 </td></tr><tr><td> Anti-Maus-HY TCR </td><td> eBioscience </td><td> XX-9930-YY * </td><td> Clone T3.70 </td></tr><tr><td> Anti-Maus-CD4 </td><td> eBioscience </td><td> XX-0042-YY * </td><td> Clone RM4-5 </td></tr><tr><td> Anti-Maus CD8a </td><td> eBioscience </td><td> XX-0081-YY * </td><td> Clone 53-6,7 </td></tr><tr><td> Anti-Maus-CD24 </td><td> eBioscience </td><td> XX-0242-YY * </td><td> Clone M1/69 </td></tr><tr><td> Anti-Maus-TCRβ </td><td> eBioscience </td><td> XX-5961-YY * </td><td> Clone H57-597 </td></tr><tr><td> Anti-Maus-CD69 Biotinylierte </td><td> eBioscience </td><td> 13-0691-YY * </td><td> Clone H1.2F3 </td></tr><tr><td> Anti-Maus-CD5 Biotinylierte </td><td> eBioscience </td><td> 13-0051-YY * </td><td> Clone 53-7,3 </td></tr><tr><td> Streptavidin </td><td> eBioscience </td><td> XX-4217-YY * </td><td></td></tr><tr><td> Durchflusszytometer </td><td> BD Biosciences - FACS Canto </td><td> 338962 </td><td></td></tr><tr><td> FACS-Röhrchen </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> Durchflusszytometrie-Analyse-Software </td><td> TreeStar - FlowJo </td><td> FlowJo v7 / 9 </td><td></td></tr><tr><td> HyClone RPMI - 1640 Medium </td><td> Thermo Scientific </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * XX variiert je nach Fluorochrom und YY variiert je nach Gefäß Größe. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Ein gesundes Immunsystem verlangt, dass T-Zellen fremde Antigene reagieren, während verbleibenden tolerant gegenüber Selbst-Antigenen. Random Umlagerung der T-Zell-Rezeptor (TCR) α und β loci erzeugt eine T-Zell-Repertoire mit riesigen Vielfalt in Antigenspezifität, sowohl sich selbst als auch ausländische. Auswahl des Repertoires während der Entwicklung im Thymus ist kritisch für die Erzeugung und sichere nützlich T-Zellen. Defekte im Thymus Auswahl zur Entwicklung von Autoimmun-und Immunschwäche-Erkrankungen 1-4. T-Zell-Vorläufer geben den Thymus als doppelt negative (DN) Thymozyten, die nicht exprimieren CD4 oder CD8 Co-Rezeptoren. Expression des αβTCR und beide Co-Rezeptoren tritt bei der doppelt positiven (DP) Bühne. Interaktion des αβTCR mit Selbst-Peptid-MHC (pMHC) durch Thymus-Zellen präsentiert bestimmt das Schicksal der DP Thymozyten. Hochaffine Wechselwirkungen zu negativen Selektion und Eliminierung führention von selbst-reaktiven Thymozyten. Low Affinitätswechselwirkungen Ergebnis in positiver Selektion und Entwicklung von CD4 oder CD8 einzigen positiven (SP) T-Zellen in der Lage zu erkennen fremde Antigene durch Selbst-MHC 5 dargestellt. Positive Selektion kann in Mäusen mit einem polyklonalen (Wildtyp) TCR Repertoires werden durch Beobachten der Erzeugung von reifen T-Zellen untersucht. Sie sind jedoch nicht ideal für das Studium der negativen Selektion, die Deletion von antigenspezifischen kleinen Populationen umfasst. Viele Modellsysteme verwendet worden, um negative Selektion zu studieren, aber variieren in ihrer Fähigkeit zu rekapitulieren physiologische Ereignisse 6. Zum Beispiel in-vitro-Stimulation von Thymozyten fehlt die Thymus-Umgebung, die innig in Auswahl beteiligt ist, während Verabreichung von exogenen Antigens an nicht-spezifische Deletion von Thymozyten 7-9 führen kann. Derzeit sind die besten Werkzeuge für die Untersuchung in vivo negative Selektion Mäuse, die eine transg ausdrückenenic TCR spezifisch für endogene Selbst-Antigen. Allerdings sind viele klassischen TCR-transgene Modelle von vorzeitigem Expression des transgenen TCRα Kette am DN Stufe dadurch, was zu einem vorzeitigen negative Selektion. Unser Labor hat den HY cd4 Modell, bei dem der transgene HY TCRα bedingt am DP Stufe exprimiert wird entwickelt, wodurch negative Selektion während der DP bis SP Übergang auftreten wie dies bei Wildtyp-Mäusen 10. Hier beschreiben wir eine Durchflusszytometrie-basiertes Protokoll zum Thymus positive und negative Selektion in der HY cd4 Mausmodell zu untersuchen. Während negative Auswahl HY cd4 Mäusen ist höchst physiologischen können diese Verfahren auch auf andere TCR-transgene Modelle angewendet werden. Wir werden außerdem allgemeine Strategien zur Analyse positive Selektion in einem polyklonalen Repertoire für alle genetisch manipulierten Mäusen.

In physiologischen TCR transgene Modelle und WT Mäusen, beginnt positive Selektion an der DP hellen Bühne, bevor er in den DP langweilig Stufe nach Antigen Begegnung. DP langweilig Thymozyten und geben Sie dann eine Übergangsregelung CD4 + CD8 lo Bühne, bevor er CD4SP oder CD8SP Thymozyten (Abbildung 2B). Mature SP Thymozyten zeichnen sich durch hohe TCR-Expression und den Verlust von CD24 (2C) gekennzeichnet. Während die CD8 d...

Watch this Scientific Journal Video about Examination of Thymic Positive and Negative Selection by Flow Cytometry at JoVE.com

Examination of Thymic Positive and Negative Selection by Flow Cytometry (Video) | JoVE

Wir präsentieren eine Durchflusszytometrie-basierte Methode zur T-Zell-Entwicklung zu untersuchen<em&gt; In vivo</em&gt; Mit genetisch manipulierten Mäusen auf einer Wildtyp-oder T-Zell-Rezeptor transgenen Hintergrund.

Prüfung Thymic positive und negative Selektion mittels Durchflusszytometrie

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Immunology and Infection

Examination of Thymic Positive and Negative Selection by Flow Cytometry

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In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry (Video) | JoVE

In-vivo-Bewertung der Nager Plasmodium Parasitämie und Merozoite Invasion durch Durchflusszytometrie

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry (Video) | JoVE

Mess Bindung und Internalisierung von Influenza A-Virus in A549-Zellen mittels Durchflusszytometrie

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry (Video) | JoVE

Phänotypische Charakterisierung von Makrophagen aus Rattenniere mittels Durchflusszytometrie

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow (Video) | JoVE

Fluoreszenz-aktivierte Zellsortierung zur Reinigung von Plasmazytoide dendritischer Zellen aus dem Knochenmark der Maus

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages (Video) | JoVE

Isolation und In-vitro- Kultur von murinen und menschlichen alveoläre Makrophagen

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis (Video) | JoVE

Charakterisierung der menschliche Monocyte Teilmengen von Vollblut Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...