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

Os autores gostariam de agradecer Bing Zhang por sua assistência técnica. Este trabalho foi financiado pelos Institutos Canadenses de Pesquisa em Saúde (MOP-86595). TAB é um CIHR novo investigador e estudioso AHFMR. QH é apoiado por uma Bolsa de Pós-Graduação CIHR Canadá - Doutorado e um Studentship tempo inteiro AIHS. SAN é apoiado por uma Rainha Elizabeth II de Bolsas de Estudo de Pós-Graduação. AYWS é apoiado por uma Bolsa de Pós-Graduação NSERC - Doutorado.

Consulte a Figura 1 para um esquema geral do protocolo experimental. 1. Dissecação <ol><li> Coloque tela de malha de aço estéril em 60 15 mm x placa de Petri. Uma unidade é necessária por amostra de tecido. </li><li> Adicionar 5 ml de solução salina equilibrada de Hank (HBSS) para cada prato. Mantenha pratos no gelo. </li><li> Euthanize ratos com CO 2. </li><li> Rato seguro para a superfície, dissecção lado ventral para cima. Pulverização do rato co...

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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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O protocolo aqui apresentado pode ser usado para examinar a selecção positiva e negativa, em não-transgénicos TCR e ratinhos transgénicos TCR. Este protocolo descreve a coloração de antigenes de superfície. Para uma análise mais aprofundada dos mecanismos moleculares, é muitas vezes necessário efectuar a coloração intracelular. Nós usamos o BD Biosciences Kit Cytofix / Cytoperm para a maioria das proteínas intracelulares e da BD Biosciences kit de coloração Foxp3 para fatores de transcrição. Nós gera...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> HyClone solução salina balanceada de Hank </td><td> Thermo Scientific </td><td> SH30030.02 </td><td></td></tr><tr><td> Telas de malha de metal </td><td> Cedarlane </td><td> CX-0080-E-01 </td><td></td></tr><tr><td> Placas de Petri (60 x 15 mm) </td><td> Fisher Scientific </td><td> 877221 </td><td></td></tr><tr><td> Seringas (3 ml) </td><td> BD Biosciences </td><td> 309657 </td><td></td></tr><tr><td> Tubos cônicos (15 ml) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> Microscópio </td><td> Zeiss - Primo Star </td><td> 415.500-00XX-000 </td><td></td></tr><tr><td> Hemocitômetro </td><td> HausserCientífico </td><td> 3110 </td><td></td></tr><tr><td> Placa de 96 poços </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Pipeta multicanal </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> Soro fetal de vitela </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> Salina tamponada com fosfato </td><td> Fisher Scientific </td><td> SH3025802 </td><td></td></tr><tr><td> A azida de sódio </td><td> IT Baker Chemical Co. </td><td> V015-05 </td><td></td></tr><tr><td> Reagente de bloqueio FcR </td><td></td><td></td><td> Clone 2.4G2 </td></tr><tr><td> Anti-rato HY TCR </td><td> eBioscience </td><td> XX-9930-AA * </td><td> Clone T3.70 </td></tr><tr><td> Anti-CD4 de ratinho </td><td> eBioscience </td><td> XX-0042-AA * </td><td> Clone RM4-5 </td></tr><tr><td> Anti-rato CD8u </td><td> eBioscience </td><td> XX-0081-AA * </td><td> Clone 53-6,7 </td></tr><tr><td> Anti-mouse CD24 </td><td> eBioscience </td><td> XX-0242-AA * </td><td> Clone M1/69 </td></tr><tr><td> Anti-rato TCRβ </td><td> eBioscience </td><td> XX-5961-AA * </td><td> Clone H57-597 </td></tr><tr><td> Anti-CD69 rato biotinilado </td><td> eBioscience </td><td> 13-0691-AA * </td><td> Clone H1.2F3 </td></tr><tr><td> Anti-rato CD5 biotinilado </td><td> eBioscience </td><td> 13-0051-AA * </td><td> Clone 53-7,3 </td></tr><tr><td> Estreptavidina </td><td> eBioscience </td><td> XX-4217-AA * </td><td></td></tr><tr><td> Citómetro </td><td> BD Biosciences - FACS Canto </td><td> 338962 </td><td></td></tr><tr><td> FACS tubos </td><td> BD Biosciences </td><td> 352052 </td><td></td></tr><tr><td> Fluxo de software de análise de citometria </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> * XX varia de acordo com fluorocromo e YY varia com o tamanho do frasco. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

Um sistema imunitário saudável requer que as células T respondem a antigénios estranhos, permanecendo tolerante para auto-antígenos. Rearranjo aleatório do receptor de células T (TCR) e loci α β gera um repertório de células T com grande diversidade de especificidade antigénica, tanto a auto-e estrangeiras. A selecção do repertório durante o desenvolvimento do timo é crítico para a geração segura e úteis células T. Defeitos de selecção tímica contribuir para o desenvolvimento de doenças auto-imunes e da imunodeficiência 1-4. Progenitores de células T do timo entrar como duplamente negativos (DN) timócitos que não expressam CD4 ou CD8 co-receptores. Expressão da αβTCR e ambos os co-receptores ocorre na fase dupla (DP) positivo. Interacção do αβTCR com auto-péptido-MHC (pMHC) apresentado pelas células do timo determina o destino do timócito DP. Interações de alta afinidade levar a seleção negativa e eliminaçãoção de auto-reactivos timócitos. Baixa afinidade interacções resultam na selecção positiva e desenvolvimento de CD4 ou CD8 positivas únicas células (SP) T capazes de reconhecer antigénios estranhos apresentados pelo MHC próprio 5. Selecção positiva pode ser estudada em ratinhos com um anticorpo policlonal repertório de TCR (tipo selvagem), observando a geração de células T maduras. No entanto, eles não são ideais para o estudo de selecção negativa, o que envolve a eliminação de pequenas populações específicas de antigénio. Muitos sistemas de modelos têm sido utilizados para estudar a selecção negativa, mas variam na sua capacidade para recapitular eventos fisiológicos 6. Por exemplo, a estimulação in vitro de timócitos falta o ambiente do timo, que está intimamente envolvido na selecção, ao passo que a administração de antigénio exógeno pode levar à supressão não específica de timócitos 7-9. Atualmente, as melhores ferramentas para estudar na seleção negativa vivo são ratos que expressam uma transgenic específica TCR para endógena antígeno próprio. No entanto, muitos modelos clássicos TCR transgénicos são caracterizados pela expressão prematura da cadeia TCRα transgénico no estádio de DN, resultando em prematuro de selecção negativa. O nosso laboratório desenvolveu o HY modelo CD4, no qual a transgénico HY TCRα condicionalmente está expresso na fase de DP, permitindo a selecção negativa para ocorrer durante a transição para DP SP como ocorre em ratinhos de tipo selvagem 10. Aqui, nós descrevemos um protocolo de citometria de fluxo com base em examinar selecção positiva e negativa do timo no modelo do rato HY CD4. Enquanto a selecção negativa nos ratos CD4 HY é altamente fisiológico, estes métodos podem também ser aplicados a outros modelos de TCR transgénicas. Nós também irá apresentar as estratégias gerais para a análise de seleção positiva em um repertório policlonal aplicáveis ​​a quaisquer camundongos geneticamente manipulados.

Em fisiológicas modelos TCR transgênicos e camundongos WT, a seleção positiva começa na fase DP brilhante antes de passar à fase DP aborrecido após encontro antígeno. Timócitos DP maçantes em seguida, digite um CD4 + fase de transição CD8 lo antes de se tornar CD4SP ou timócitos CD8SP (Figura 2B). Timócitos maduros SP são caracterizados pela expressão de TCR e da elevada perda de CD24 (Figura 2C). Enquanto o CD8 por ...

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

Apresenta-se um método de citometria de fluxo com base para examinar o desenvolvimento das células T<em&gt; In vivo</em&gt; Usando camundongos geneticamente manipulados em um tipo selvagem ou de fundo do receptor de células T transgênico.

Exame de seleção positiva e negativa Tímico por citometria de fluxo

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

Medindo Anexo e Internalização do Vírus da Influenza A em células A549 por citometria de fluxo

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

Caracterização fenotípica dos macrófagos a partir de rim de rato por Citometria de Fluxo

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

Classificação de células activadas por fluorescência Para a purificação de células dendríticas plasmocitï¿½des do rato de Medula Óssea

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 cultura In Vitro de macrófagos alveolares murino e humanos

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

Caracterização de subconjuntos de monócitos humanos por sangue total Flow Cytometry Analysis

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