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

लेखकों को अपने तकनीकी सहायता के लिए बिंग जांग धन्यवाद देना चाहूंगा. यह काम स्वास्थ्य अनुसंधान (एमओपी 86,595) के लिए कनाडा के संस्थानों द्वारा वित्त पोषित किया गया था. TAB एक CIHR नई अन्वेषक और AHFMR विद्वान है. डॉक्टरेट और एक AIHS छात्रवृत्ति पूर्णकालिक QH - एक CIHR कनाडा ग्रेजुएट छात्रवृत्ति के द्वारा समर्थित है. SAN एक महारानी एलिजाबेथ द्वितीय ग्रेजुएट छात्रवृत्ति द्वारा समर्थित है. डॉक्टरेट - AYWS एक NSERC स्नातकोत्तर छात्रवृत्ति द्वारा समर्थित है.

प्रयोगात्मक प्रोटोकॉल के एक समग्र योजना के लिए आकृति 1 देखें. 1. विच्छेदन <ol><li> 60 x 15 मिमी पेट्री डिश में बाँझ इस्पात जाल स्क्रीन. एक ऊतक का नमूना प्रति यूनिट की जरूरत है. </li><li> प्रत्येक पकवान ?...

<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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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. K., Cado, D., Winoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+orphan+steroid+receptor+Nur77+in+apoptosis+accompanying+antigen-induced+negative+selection.">A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection.</a> Immunity. 3, 273-282 (1995).</li><li>Zhou, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Nur77/Nurr1+leads+to+inefficient+clonal+deletion+of+self-reactive+T+cells.">Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells.</a> J. Exp. Med. 183, 1879-1892 (1996).</li><li>Baldwin, T. A., 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=Transcriptional+analysis+of+clonal+deletion+in+vivo.">Transcriptional analysis of clonal deletion in vivo.</a> J. Immunol. 179, 837-844 (2007).</li></ol>

यहाँ प्रस्तुत प्रोटोकॉल गैर TCR ट्रांसजेनिक और TCR ट्रांसजेनिक चूहों में सकारात्मक और नकारात्मक चयन की जांच करने के लिए इस्तेमाल किया जा सकता है. इस प्रोटोकॉल की सतह प्रतिजनों के धुंधला का वर्णन करता है. ?...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक) </td></tr><tr><td> HyClone हांक संतुलित नमक समाधान </td><td> थर्मो वैज्ञानिक </td><td> SH30030.02 </td><td></td></tr><tr><td> धातु जाल स्क्रीन </td><td> Cedarlane </td><td> CX-0,080-E-01 </td><td></td></tr><tr><td> पेट्री डिश (60 x 15 मिमी) </td><td> फिशर साइंटिफिक </td><td> 877221 </td><td></td></tr><tr><td> सीरिंज (3 मिलीलीटर) </td><td> बी.डी. बायोसाइंसेज </td><td> 309657 </td><td></td></tr><tr><td> शंक्वाकार ट्यूब (15 मिलीलीटर) </td><td> Sarstedt </td><td> 62.554.205 </td><td></td></tr><tr><td> माइक्रोस्कोप </td><td> Zeiss - Primo स्टार </td><td> 415,500 00XX-000 </td><td></td></tr><tr><td> Hemocytometer </td><td> HAUSSERवैज्ञानिक </td><td> 3110 </td><td></td></tr><tr><td> 96 अच्छी तरह से थाली </td><td> Sarstedt </td><td> 82.1582.001 </td><td></td></tr><tr><td> Multichannel विंदुक </td><td> Fisherbrand </td><td> 21-377-829 </td><td></td></tr><tr><td> भ्रूण बछड़ा सीरम </td><td> PAA </td><td> A15-701 </td><td></td></tr><tr><td> फास्फेट खारा buffered </td><td> फिशर साइंटिफिक </td><td> SH3025802 </td><td></td></tr><tr><td> सोडियम azide </td><td> आईटी बेकर रासायनिक कं </td><td> V015-05 </td><td></td></tr><tr><td> FCR अवरुद्ध अभिकर्मक </td><td></td><td></td><td> क्लोन 2.4G2 </td></tr><tr><td> विरोधी माउस हरियाणा TCR </td><td> eBioscience </td><td> XX-9930-YY * </td><td> क्लोन T3.70 </td></tr><tr><td> विरोधी माउस CD4 </td><td> eBioscience </td><td> XX-0042-YY * </td><td> RM4-5 क्लोन </td></tr><tr><td> एकnti-माउस CD8α </td><td> eBioscience </td><td> XX-0081-YY * </td><td> 53-6.7 क्लोन </td></tr><tr><td> CD24 विरोधी माउस </td><td> eBioscience </td><td> XX-0,242 YY * </td><td> M1/69 क्लोन </td></tr><tr><td> विरोधी माउस TCRβ </td><td> eBioscience </td><td> XX-5961-YY * </td><td> H57-597 क्लोन </td></tr><tr><td> CD69 विरोधी माउस Biotinylated </td><td> eBioscience </td><td> 13-0691 YY * </td><td> क्लोन H1.2F3 </td></tr><tr><td> CD5 विरोधी माउस Biotinylated </td><td> eBioscience </td><td> 13-0051 YY * </td><td> 53-7.3 क्लोन </td></tr><tr><td> Streptavidin </td><td> eBioscience </td><td> XX-4217-YY * </td><td></td></tr><tr><td> प्रवाह cytometer </td><td> बी.डी. बायोसाइंसेज FACS सर्ग </td><td> 338962 </td><td></td></tr><tr><td> FACS ट्यूबों </td><td> बी.डी. बायोसाइंसेज </td><td> 352052 </td><td></td></tr><tr><td> प्रवाह cytometry विश्लेषण सॉफ्टवेयर </td><td> TreeStar - Flowjo </td><td> 9 / v7 FlowJo </td><td></td></tr><tr><td> 1640 मध्यम HyClone RPMI </td><td> थर्मो वैज्ञानिक </td><td> SH30027.01 </td><td></td></tr><tr><td></td><td></td><td></td><td> * XX बदलता है द्वारा fluorochrome और YY शीशी आकार से भिन्न होता है. </td></tr></tbody></table>

examination of thymic positive and negative selection by flow cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

शारीरिक TCR ट्रांसजेनिक मॉडल और WT चूहों में सकारात्मक चयन प्रतिजन मुठभेड़ के बाद डी पी सुस्त चरण में चलती से पहले डी पी उज्ज्वल चरण में शुरू होता है. डी पी सुस्त thymocytes तो एक संक्रमणकालीन CD4 + CD...

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

हम एक प्रवाह cytometry आधारित टी सेल के विकास की जांच के लिए विधि मौजूद<em&gt; Vivo में</em&gt; एक wildtype या टी सेल रिसेप्टर ट्रांसजेनिक पृष्ठभूमि पर आनुवंशिक चालाकी से चूहों का उपयोग.

फ्लो Thymic सकारात्मक और नकारात्मक चयन परीक्षा

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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Immunology and Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

फिक्स्ड और जीवित कोशिकाओं में प्रवाह cytometry द्वारा Calpain गतिविधि को मापने

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

मानवीय इन विट्रो में इम्यून असंतुलन का एक प्रारंभिक राज्य की मान्यता के लिए स्क्रीनिंग उपकरण के रूप में दमन

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

की बातचीत का अध्ययन<em&gt; स्ताफ्य्लोकोच्चुस</em&gt; फ्लो और समय चूक माइक्रोस्कोपी द्वारा न्यूट्रोफिल साथ

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

फ्लो द्वारा प्राथमिक प्रतिरक्षा सेल subpopulations साथ फ्लोरोसेंट Nanoparticle बातचीत का पता लगाने

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

फ्लो द्वारा कृंतक प्लाज्मोडियम पेरासाइटिमिया के vivo आकलन और Merozoite आक्रमण में

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

फ्लो द्वारा A549 कोशिकाओं में लगाव और Internalization इन्फ्लूएंजा के एक वायरस को मापने

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

से फ्लो द्वारा चूहे गुर्दे से मैक्रोफेज की प्ररूपी विशेषता

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

माउस अस्थि मज्जा से Plasmacytoid वृक्ष के समान कोशिकाओं की शुद्धि के लिए प्रतिदीप्ति सक्रिय सेल छंटनी

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

अलगाव और इन विट्रो संस्कृति Murine और मानव वायुकोशीय मैक्रोफेज

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

पूरे रक्त प्रवाह Cytometry विश्लेषण द्वारा मानव Monocyte सबसेट का लक्षण वर्णन

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