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

Gli autori desiderano ringraziare Bing Zhang per la sua assistenza tecnica. Questo lavoro è stato finanziato dal Canadian Institutes for Health Research (MOP-86595). TAB è un investigatore CIHR Nuovo e Scholar AHFMR. QH è sostenuto da una borsa di studio CIHR Canada Graduate - Dottorato e un AIHS tempo pieno Studentship. SAN è sostenuto da una borsa di studio Queen Elizabeth II Graduate. AYWS è sostenuto da una borsa di studio post-laurea NSERC - Dottorato.

Fare riferimento alla Figura 1 per uno schema generale del protocollo sperimentale. 1. Dissezione <ol><li> Luogo sterile setaccio a maglia d&#39;acciaio in 60 x 15 millimetri capsula di Petri. Una unità è necessaria per campione di tessuto. </li><li> Aggiungere 5 ml di soluzione salina bilanciata di Hank (HBSS) a ogni piatto. Tenere i piatti sul ghiaccio. </li><li> Eutanasia topi con CO 2. </li><li> Del mouse sulla superficie protetta dissezione, lato ventrale...

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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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Immunol. 21, 139-176 (2003).</li><li>McCaughtry, T. M., 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=Central+tolerance:+what+have+we+learned+from+mice.">Central tolerance: what have we learned from mice.</a> Seminars in immunopathology. 30, 399-409 (2008).</li><li>Zhan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Without+peripheral+interference,+thymic+deletion+is+mediated+in+a+cohort+of+double-positive+cells+without+classical+activation.">Without peripheral interference, thymic deletion is mediated in a cohort of double-positive cells without classical activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 1197-1202 (2003).</li><li>Brewer, J. A., Kanagawa, O., Sleckman, B. P., Muglia, L. 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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. Med. 200, 493-505 (2004).</li><li>Saini, M. <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+Zap70+expression+during+thymocyte+development+enables+temporal+separation+of+CD4+and+CD8+repertoire+selection+at+different+signaling+thresholds.">Regulation of Zap70 expression during thymocyte development enables temporal separation of CD4 and CD8 repertoire selection at different signaling thresholds.</a> Science signaling. 3, ra23 (2010).</li><li>Hu, Q., Sader, A., Parkman, J. C., 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=Bim-mediated+apoptosis+is+not+necessary+for+thymic+negative+selection+to+ubiquitous+self-antigens.">Bim-mediated apoptosis is not necessary for thymic negative selection to ubiquitous self-antigens.</a> J. 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. 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Il protocollo qui presentato può essere utilizzato per esaminare selezione positiva e negativa in non-transgenici TCR e TCR topi transgenici. Questo protocollo descrive la colorazione degli antigeni di superficie. Per un&#39;ulteriore analisi dei meccanismi molecolari, è spesso necessario eseguire la colorazione intracellulare. Usiamo il BD Biosciences Cytofix / Cytoperm Kit per la maggior parte delle proteine ​​intracellulari e la Foxp3 BD Biosciences Kit di colorazione per fattori di trascrizione. Noi di solito ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Sale HyClone Hank soluzione equilibrata </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Rete metallica schermi </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Piastre di Petri (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877221 </td><td></td></tr><tr><td> Siringhe (3 ml) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> Provette coniche (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Microscopio </td><td> Zeiss - Primo Star </td><td> 415500-00XX-000 </td><td></td></tr><tr><td> Emocitometro </td><td> HausserScientifico </td><td> 3110 </td><td></td></tr><tr><td> Piastra a 96 pozzetti </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Pipetta multicanale </td><td> FISHERBRAND </td><td> 21-377-829 </td><td></td></tr><tr><td> Siero fetale di vitello </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Tampone fosfato salino </td><td> Fisher Scientific </td><td> SH3025802 </td><td></td></tr><tr><td> Sodio azide </td><td> IT Baker Chemical Co. </td><td> V015-05 </td><td></td></tr><tr><td> FcR reagente bloccante </td><td></td><td></td><td> Clone 2.4G2 </td></tr><tr><td> Anti-mouse HY TCR </td><td> eBioscience </td><td> XX-9930-AA * </td><td> Clone T3.70 </td></tr><tr><td> Anti-topo CD4 </td><td> eBioscience </td><td> XX-0042-AA * </td><td> Clone RM4-5 </td></tr><tr><td> Lanti-mouse CD8α </td><td> eBioscience </td><td> XX-0081-AA * </td><td> Clone 53-6,7 </td></tr><tr><td> Anti-topo CD24 </td><td> eBioscience </td><td> XX-0242-AA * </td><td> Clone M1/69 </td></tr><tr><td> Anti-mouse TCRβ </td><td> eBioscience </td><td> XX-5961-AA * </td><td> Clone H57-597 </td></tr><tr><td> Anti-topo CD69 Biotinylated </td><td> eBioscience </td><td> 13-0691-AA * </td><td> Clone H1.2F3 </td></tr><tr><td> Anti-mouse CD5 Biotinylated </td><td> eBioscience </td><td> 13-0051-AA * </td><td> Clone 53-7,3 </td></tr><tr><td> Streptavidina </td><td> eBioscience </td><td> XX-4217-AA * </td><td></td></tr><tr><td> Citometro di flusso </td><td> BD Biosciences - FACS Canto </td><td> 338962 </td><td></td></tr><tr><td> FACS tubi </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> Citometria di flusso software di analisi </td><td> TreeStar - Flowjo </td><td> FlowJo v7 / 9 </td><td></td></tr><tr><td> HyClone RPMI - 1640 </td><td> Thermo Scientific </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * Varia da XX e YY fluorocromo varia per grandezza flaconcino. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Un sistema immunitario sano è necessario che le cellule T rispondono agli antigeni estranei, pur rimanendo tolleranza di auto-antigeni. Riarrangiamento casuale del recettore delle cellule T (TCR) α e β loci genera un repertorio T cella con grande diversità nella specificità antigenica, sia per sé ed esteri. Selezione del repertorio durante lo sviluppo nel timo è critica per generare le cellule T sicuro e utile. Difetti di selezione timica contribuire allo sviluppo di malattie autoimmuni e immunodeficienza 1-4. Progenitori delle cellule T inserire il timo come doppi negativi (DN) timociti che non esprimono CD4 o CD8 co-recettori. Espressione del αβTCR ed entrambi co-recettori avviene a doppio positivo (DP) stadio. Interazione del αβTCR con auto-peptide-MHC (pMHC) presentata da parte delle cellule del timo determina il destino del timociti DP. Interazioni ad alta affinità portare alla selezione negativa e di eliminazionezione di auto-reattivi timociti. Bassa affinità risultato interazioni in selezione positiva e sviluppo di CD4 o CD8 singoli positivi (SP) cellule T capaci di riconoscere antigeni estranei presentati da auto-MHC 5. Selezione positiva può essere studiata in topi con un anticorpo (tipo selvatico) repertorio TCR osservando la generazione di cellule T mature. Tuttavia, essi non sono ideali per lo studio di selezione negativa, che comporta l&#39;eliminazione di piccoli antigene-specifiche popolazioni. Molti sistemi modello sono stati utilizzati per studiare selezione negativa, ma variano nella loro capacità di ricapitolare eventi fisiologici 6. Per esempio, stimolazione in vitro dei timociti manca dell&#39;ambiente timica che è intimamente coinvolto nella selezione, mentre la somministrazione di antigene esogeno può portare a non-specifico delezione di timociti 7-9. Attualmente, i migliori strumenti per lo studio in vivo selezione negativa sono topi che esprimono una transgENIC specifico TCR per endogena auto-antigene. Tuttavia, molti classici modelli transgenici TCR sono caratterizzati dalla prematura espressione della catena transgenico TCRα nella fase DN, con conseguente prematuro selezione negativa. Il nostro laboratorio ha sviluppato il modello HY cd4, in cui il transgenico HY TCRα condizionatamente espressa nella fase DP, consentendo la selezione negativa a verificarsi durante il DP di transizione SP come avviene nei topi di tipo selvatico 10. Qui, si descrive un protocollo basato citometria di flusso per esaminare timica selezione positiva e negativa nel topo HY modello CD4. Mentre la selezione negativa in topi CD4 HY è altamente fisiologica, questi metodi possono essere applicati anche ad altri modelli transgenici TCR. Ci sarà anche la presentazione delle strategie generali per l&#39;analisi di selezione positiva in un repertorio policlonale applicabile a tutti i topi geneticamente manipolati.

In fisiologiche TCR modelli transgenici e topi WT, selezione positiva inizia nella fase DP luminoso prima di passare alla fase di DP opaca dopo l&#39;incontro di antigene. Timociti DP opaco quindi inserire una transizione CD4 + CD8 lo stadio prima di diventare CD4SP o timociti CD8SP (Figura 2B). Timociti maturi SP sono caratterizzati da elevata espressione TCR e perdita di CD24 (Figura 2C). Mentre il CD8 da CD4 profilo può rivelare...

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

Vi presentiamo un metodo basato citometria di flusso per esaminare lo sviluppo delle cellule T<em&gt; In vivo</em&gt; Utilizzando topi geneticamente manipolati su un tipo selvatico o T sfondo recettore delle cellule transgeniche.

Esame di Selezione timica positivi e negativi per Citometria a Flusso

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

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Caratterizzazione fenotipica dei macrofagi dal rene di ratto mediante citometria di flusso

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

Separazione delle cellule fluorescenza-attivato per la purificazione delle cellule dendritiche plasmacitoidi dal midollo osseo di topo

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

Isolamento e coltura In Vitro dei macrofagi alveolari murini ed umani

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

Caratterizzazione dei sottoinsiemi di monociti umani da sangue intero flusso Cytometry analisi

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