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

Författarna vill tacka Bing Zhang för hans tekniskt bistånd. Detta arbete har finansierats av den kanadensiska Institutes for Health Research (MOP-86.595). TAB är en CIHR ny utredare och AHFMR Scholar. QH stöds av en CIHR Canada Graduate Scholarship - Utbildning och AIHS Heltid STUDENTTILLVARO. SAN stöds av en drottning Elizabeth II Graduate Scholarship. AYWS stöds av en NSERC Utbildningsbidrag - Utbildning.

Se figur 1 för en övergripande ordning av den experimentella protokollet. 1. Dissektion <ol><li> Placera steril stålnät skärmen i 60 x 15 mm petriskål. En enhet behövs per vävnadsprov. </li><li> Tillsätt 5 ml av Hanks balanserade saltlösning (HBSS) till varje skål. Håll rätter på is. </li><li> Euthanize möss med CO 2. </li><li> Säker musen för att dissektion yta, ventrala sidan uppåt. Sprej mus med 70% etanol för sterilisering och för att sä...

<ol>
	<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. 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Protokollet som presenteras här kan användas för att undersöka positiv och negativ selektion i icke-TCR transgena och TCR-transgena möss. Detta protokoll beskriver färgning av ytantigener. För ytterligare analys av molekylära mekanismer, är det ofta nödvändigt att utföra intracellulär färgning. Vi använder BD Biosciences Cytofix / Cytoperm Kit för de flesta intracellulära proteiner och BD Biosciences Foxp3 färgningskit för transkriptionsfaktorer. Vi förvärvar brukar våra prover direkt efter färgni...

<table border="1"><tbody><tr><td> Namnet av reagenset </td><td> Företag </td><td> Katalognummer </td><td> Kommentarer (tillval) </td></tr><tr><td> HyClone Hanks balanserade saltlösning </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Metal mesh </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Petriskålar (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877.221 </td><td></td></tr><tr><td> Sprutor (3 ml) </td><td> BD Biosciences </td><td> 309.657 </td><td></td></tr><tr><td> Koniska rör (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> HausserVetenskaplig </td><td> 3110 </td><td></td></tr><tr><td> 96-brunnars platta </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Multikanalpipett </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> Fetalt kalvserum </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Fosfatbuffrad saltlösning </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 blockeringsreagens </td><td></td><td></td><td> Klon 2.4G2 </td></tr><tr><td> Anti-mus HY TCR </td><td> eBioscience </td><td> XX-9930-YY * </td><td> Klon T3.70 </td></tr><tr><td> Anti-mus CD4 </td><td> eBioscience </td><td> XX-0042-YY * </td><td> Klon RM4-5 </td></tr><tr><td> ENNTI-mus CD8a </td><td> eBioscience </td><td> XX-0081-YY * </td><td> Klon 53-6,7 </td></tr><tr><td> Anti-mus CD24 </td><td> eBioscience </td><td> XX-0242-YY * </td><td> Klon M1/69 </td></tr><tr><td> Anti-mus TCRβ </td><td> eBioscience </td><td> XX-5961-YY * </td><td> Klon H57-597 </td></tr><tr><td> Anti-mus CD69 Biotinylerad </td><td> eBioscience </td><td> 13-0691-ÅÅ * </td><td> Klon H1.2F3 </td></tr><tr><td> Anti-mus CD5 Biotinylerad </td><td> eBioscience </td><td> 13-0051-ÅÅ * </td><td> Klon 53-7,3 </td></tr><tr><td> Streptavidin </td><td> eBioscience </td><td> XX-4217-YY * </td><td></td></tr><tr><td> Flödescytometern </td><td> BD Biosciences - FACS Canto </td><td> 338.962 </td><td></td></tr><tr><td> FACS-rör </td><td> BD Biosciences </td><td> 352.052 </td><td></td></tr><tr><td> Flödescytometrianalys programvara </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 varierar beroende fluorokromen och YY varierar med flaskan storlek. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Ett friskt immunsystem kräver att T-celler svarar på främmande antigener samtidigt som den är tolerant mot själv-antigener. Slumpvis omlagring av T-cell receptorn (TCR) α och β loci genererar en T-cell repertoar med stor mångfald i antigenspecificitet, både för sig själv och utländska. Val av repertoaren under utveckling i tymus är kritisk för att generera säkra och användbara T-celler. Defekter i tymus val bidra till utvecklingen av autoimmuna och immunbristsjukdomar 1-4. T-cell stamfäder in tymus som dubbla negativa (DN) tymocyter som inte uttrycker CD4 eller CD8 co-receptorer. Redovisning av αβTCR och båda co-receptorer sker vid dubbla positiva (DP) steg. Interaktion av αβTCR med själv-peptid-MHC (pMHC) presenteras av tymusceller bestämmer ödet för DP tymocyt. Hög affinitet interaktioner leder till negativ selektion och elimineringning av självreaktiva tymocyter. Låg affinitet interaktioner resulterar i positiv selektion och utveckling av CD4 eller CD8 enda positiv (SP) T-celler kan känna igen främmande antigener presenteras av själv-MHC 5. Positiv selektion kan studeras i möss med en polyklonal (vildtyp) TCR repertoar genom att observera alstring av mogna T-celler. Emellertid, är de inte ideala för studier av negativ selektion, som involverar deletion av små antigenspecifika populationer. Många modellsystem har använts för att studera negativ selektion men varierar i sin förmåga att rekapitulera fysiologiska händelser 6. Till exempel, in vitro-stimulering av tymocyter saknar tymus miljö som är intimt inblandad i valet, medan administrering av exogent antigen kan leda till icke-specifik deletion av tymocyter 7-9. För närvarande är de bästa verktygen för att studera in vivo negativ selektering är möss som uttrycker en transgENIC TCR specifik för endogen själv-antigen. Men många klassiska TCR transgena modeller som kännetecknas av tidig expression av den transgena TCRα kedjan vid DN stadiet, vilket resulterar i för tidig negativ selektion. Vårt laboratorium har utvecklat HY CD4-modellen, där transgena HY TCRα är villkorligt uttryck i DP stadiet, vilket negativt urval ske under DP SP övergång som sker i vildtyp möss 10. Här beskriver vi ett flödescytometri-baserat protokoll för att undersöka tymus positiv och negativ selektion i HY CD4 musmodellen. Medan negativ selektion i HY CD4 möss är mycket fysiologisk, kan dessa metoder även tillämpas på andra TCR transgena modeller. Vi kommer också att presentera övergripande strategier för att analysera positiv selektion i en polyklonal repertoar som gäller för alla genetiskt manipulerade möss.

I fysiologiska TCR transgena modeller och WT möss, börjar positiv selektion på DP ljusa stadiet innan du flyttar in i DP tråkig steget efter antigen möter. DP tråkig tymocyter in sedan en övergångsperiod CD4 + CD8 Lo skede innan han blev CD4SP eller CD8SP tymocyter (Figur 2B). Mogna SP tymocyter kännetecknas av hög TCR expression och förlust av CD24 (figur 2C). Medan CD8 av CD4 profil kan avslöja brister i positiv selekt...

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 presenterar ett flödescytometri-baserad metod för att undersöka T-cell-utveckling<em&gt; In vivo</em&gt; Använda genetiskt manipulerade möss på en vildtyp eller T-cell receptor transgena bakgrund.

Undersökning av thymus Positiv och negativ selektion genom flödescytometri

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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Fenotypisk karakterisering av makrofager från råttnjure med flödescytometri

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

Fluorescensaktiverad cellsortering för Rening av Plasmacytoid dendritiska celler från benmärg från mus

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

Isolering och In Vitro kultur av murina och mänskliga alveolära makrofager

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

Karakterisering av mänskliga monocyt delmängder av helblod Flow flödescytometri analys

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