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

Forfatterne vil gerne takke Bing Zhang for hans teknisk bistand. Dette arbejde blev finansieret af den canadiske Institutes for Health Research (MOP-86.595). TAB er en CIHR New Investigator og AHFMR Scholar. QH er understøttet af en CIHR Canada Graduate Scholarship - Ph.d. og en AIHS Fuldtids studentship. SAN er understøttet af en dronning Elizabeth II Graduate Scholarship. AYWS understøttes af en NSERC Postgraduate Scholarship - Doctoral.

Se figur 1 for en samlet ordning af forsøgsprotokollen. 1. Dissection <ol><li> Sted sterile stål mesh sigte i 60 x 15 mm petriskål. En enhed er nødvendig pr vævsprøve. </li><li> Der tilsættes 5 ml Hanks balancerede saltopløsning (HBSS) til hver skål. Hold retter på is. </li><li> Aflive mus med CO 2. </li><li> Sikker mus til dissektion overflade, ventral opad. Spray mus med 70% ethanol til sterilisering og sikre, at pelsen sammenfiltret ned. </li><li> A...

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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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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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Protokollen præsenteres her kan anvendes til at undersøge positiv og negativ selektion i ikke-TCR transgene og TCR-transgene mus. Denne protokol beskrives farvning af overflade-antigener. For en nærmere analyse af molekylære mekanismer, er det ofte nødvendigt at udføre intracellulær farvning. Vi bruger BD Biosciences Cytofix / Cytoperm Kit for de fleste intracellulære proteiner og BD Biosciences foxp3 farvningssæt for transkriptionsfaktorer. Vi plejer at købe vores prøver umiddelbart efter farvning. Imidlerti...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> HyClone Hanks balancerede saltopløsning </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Metalgitter skærme </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Petriskåle (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877.221 </td><td></td></tr><tr><td> Sprøjter (3 ml) </td><td> BD Biosciences </td><td> 309.657 </td><td></td></tr><tr><td> Koniske rør (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Microscope </td><td> Zeiss - Primo stjerne </td><td> 415500-00XX-000 </td><td></td></tr><tr><td> Hæmocytometer </td><td> HausserVidenskabelig </td><td> 3110 </td><td></td></tr><tr><td> Plade med 96 brønde </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Multikanalpipette </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> Føtalt kalveserum </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Phosphatpufret saltvand </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 blokeringsreagens </td><td></td><td></td><td> Klon 2.4G2 </td></tr><tr><td> Anti-muse HY TCR </td><td> eBioscience </td><td> XX-9930-YY * </td><td> Klon T3.70 </td></tr><tr><td> Anti-muse-CD4 </td><td> eBioscience </td><td> XX-0042-YY * </td><td> Klon RM4-5 </td></tr><tr><td> ANTI-muse-CD8a </td><td> eBioscience </td><td> XX-0081-YY * </td><td> Klon 53-6,7 </td></tr><tr><td> Anti-muse 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-muse CD69 biotinyleret </td><td> eBioscience </td><td> 13-0691-YY * </td><td> Klon H1.2F3 </td></tr><tr><td> Anti-mus CD5 biotinyleret </td><td> eBioscience </td><td> 13-0051-YY * </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> Flowcytometer </td><td> BD Biosciences - FACS Canto </td><td> 338.962 </td><td></td></tr><tr><td> FACS-rør </td><td> BD Biosciences </td><td> 352.052 </td><td></td></tr><tr><td> Flowcytometrianalyse 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 varierer med fluorochrom og YY varierer efter hætteglasstørrelse. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Et sundt immunsystem kræver, at T-celler reagerer på fremmede antigener, mens de resterende tolerant til selv-antigener. Tilfældig omlejring af T-cellereceptoren (TCR) α og β loci genererer et T-celle-repertoire med stor forskel på antigenspecificitet, både til sig selv og fremmede. Udvælgelse af repertoiret under udvikling i thymus er kritisk for generering af sikre og egnede T-celler. Defekter i thymisk udvælgelse bidrage til udviklingen af autoimmune og immundefekt lidelser 1-4. T-celle-progenitorer ind thymus som dobbelt-negative (DN) thymocytter, som ikke udtrykker CD4-eller CD8-co-receptorer. Ekspression af αβTCR og begge coreceptorer forekommer ved den dobbelte positive (DP) fase. Interaktion af αβTCR med selv-peptid-MHC (pMHC) præsenteret ved thymiske celler bestemmer skæbnen for DP thymocyt. Høj affinitet interaktioner fører til negativ selektion og elimineringtion af selvnedbrydende thymocytter. Lav affinitet interaktioner resulterer i positiv selektion og udvikling af CD4 eller CD8 enkelte positive (SP) T-celler stand til at genkende fremmede antigener præsenteret af selv-MHC 5. Positiv selektion kan undersøges i mus med et polyklonalt (vildtype) TCR repertoire ved at observere dannelsen af ​​modne T-celler. De er imidlertid ikke ideelle til studiet af negativ udvælgelse, der involverer deletion af små antigen-specifikke populationer. Mange modelsystemer har været anvendt til at undersøge negativ selektion, men varierer i deres evne til at rekapitulere fysiologiske begivenheder 6. For eksempel mangler in vitro-stimulering af thymocytter den thymiske miljø, som er tæt involveret i selektion, mens indgivelse af exogent antigen kan føre til ikke-specifik deletion af thymocytter 7-9. I øjeblikket er de bedste værktøjer til at studere in vivo negativ selektion er mus, der udtrykker en transgENIC TCR specifikke for endogene selv-antigen. Imidlertid er mange klassiske TCR-transgene modeller karakteriseret ved for tidlig ekspression af det transgene TCRα kæde på DN fase, hvilket resulterer i for tidlig negativ selektion. Vores laboratorium har udviklet HY CD4 model, hvor det transgene HY TCRα er betinget udtrykt på DP fase, hvilket tillader negativ selektion til at forekomme i DP til SP overgang som forekommer i vildtype-mus 10. Her beskriver vi en flowcytometri-baseret protokol til at undersøge thymisk positiv og negativ selektion i HY CD4 musemodel. Mens negativ selektion i HY CD4 mus er meget fysiologisk, kan disse fremgangsmåder også anvendes på andre TCR-transgene modeller. Vi vil også præsentere generelle strategier til at analysere positiv selektion i et polyklonalt repertoire gælder for genetisk manipulerede mus.

I fysiologiske TCR transgene modeller og WT mus, begynder positiv markering på DP lyse scene før du flytter ind i DP kedelig etape efter antigen encounter. DP kedelige thymocytter derefter indtaste et overgangs CD4 + CD8 lo etape før han blev CD4SP eller CD8SP thymocytter (figur 2B). Ældre SP thymocyter er kendetegnet ved høj TCR ekspression og tab af CD24 (figur 2C). Mens CD8 af CD4 profil kan afsløre fejl i positiv selektion...

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 præsenterer en flowcytometri-baserede metode til at undersøge T-celleudvikling<em&gt; In vivo</em&gt; Hjælp af genetisk manipulerede mus på en vildtype-eller T-cellereceptor transgen baggrund.

Undersøgelse af tymisk positiv og negativ selektion ved flowcytometri

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

Fluorescens-aktiveret cellesortering til oprensning af Plasmacytoide dendritiske celler fra knoglemarven i 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

Isolation og In Vitro kultur af Murine og menneskelige alveolær 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 af humane monocyt delmængder af fuldblod Flow flowcytometri analyse

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