Name: antigen specific in vivo killing assay using cfse labeled target cells
Uploaded: 2010-11-09T17:00:00
Description: Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Ik wil Bianca Mothe, Carla Oseroff, en Marie-France DelGuercio bedanken voor de invoering van mij om immunologische assays en muis hanteren. Werken in het lab van GA Splitter wordt gefinancierd door NIH subsidie ​​1-RO1-AI-073558, GLRCE subsidie ​​1-U54-AI-057153, en BARD VS-3829-06.

1. Uitlokken van Target-specifieke CTL&#39;s Effector door immunisatie (Day 0) <ol><li> Voordat u begint, beslissen over een specifieke effector: target-systeem. In dit voorbeeld van een klassieke in-vivo-CTL-test, zullen we gebruik maken van een gezuiverd peptide immunisatie specifieke CD8 + T-cellen te lokken. Hetzelfde peptide zal worden gebruikt om de syngene doelcellen pols, het creëren van een specifieke effector: target-systeem. Als alternatief kunt u ervoor kiezen om hele pathogeen of eiwit te gebrui...

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	<li>Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G., Ahmed, R. <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+epitope+escape+on+PD-1+expression+and+CD8+T-cell+exhaustion+during+chronic+infection.">Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection.</a> J Virol. 83 (9), 4386-4394 (2009).</li><li>Jenkins, M. R., Tsun, A., Stinchcombe, J. C., Griffiths, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+strength+of+T+cell+receptor+signal+controls+the+polarization+of+cytotoxic+machinery+to+the+immunological+synapse.">The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.</a> Immunity. 31 (4), 621-631 (2009).</li><li>Ingulli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+tolerance+and+immunity+in+vivo+by+CFSE-labeling+of+administered+cells.">Tracing tolerance and immunity in vivo by CFSE-labeling of administered cells.</a> Methods Mol Biol. 380, 365-376 (2007).</li><li>Durward, M. A., Harms, J., Magnani, D. M., Eskra, L., Splitter, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discordant+Brucella+melitensis+antigens+yield+cognate+CD8++T+cells+in+vivo.">Discordant Brucella melitensis antigens yield cognate CD8+ T cells in vivo.</a> Infect Immun. 78 (1), 168-176 (2010).</li><li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J Immunol Methods. 171 (1), 131-137 (1994).</li><li>Weston, S. A., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+dyes+for+lymphocyte+migration+studies.+Analysis+by+flow+cytometry+and+fluorescence+microscopy.">New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.</a> J Immunol Methods. 133 (1), 87-97 (1990).</li><li>Akondy, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yellow+fever+virus+vaccine+induces+a+broad+and+polyfunctional+human+memory+CD8++T+cell+response.">The yellow fever virus vaccine induces a broad and polyfunctional human memory CD8+ T cell response.</a> J Immunol. 183 (12), 7919-7930 (2009).</li><li>Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+antigen-driven+responses+by+flow+cytometry:+monitoring+proliferation+by+dye+dilution.">Tracking antigen-driven responses by flow cytometry: monitoring proliferation by dye dilution.</a> Cytometry A. 73 (11), 1019-1034 (2008).</li><li>Labadi, A., Balogh, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+preferences+in+serosal+homing+and+distribution+of+peritoneal+B-cell+subsets+revealed+by+in+situ+CFSE+labeling.">Differential preferences in serosal homing and distribution of peritoneal B-cell subsets revealed by in situ CFSE labeling.</a> Int Immunol. 21 (9), 1047-1056 (2009).</li><li>Suffner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+support+homeostatic+expansion+of+Foxp3++regulatory+T+cells+in+Foxp3.LuciDTR+mice.">Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice.</a> J Immunol. 184 (4), 1810-1820 .</li></ol>

Deze test kan worden aangepast aan de proliferatieve capaciteit van de cellen, met inbegrip klonale expansie te onderzoeken, omdat CFSE verdeelt relatief gelijkelijk over nageslacht 10,11. Het is ook mogelijk om CFSE labeling gebruiken om celmigratie 6,12 onderzoeken, maar er zijn ook andere cel tracing methoden die kunnen meer passend zijn, afhankelijk van uw hypothese, bijvoorbeeld tetrameren en bioluminescentie 13, die ook kan worden gecombineerd met CFSE labeling. <p class="jove_...

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antigen specific in vivo killing assay using cfse labeled target cells

Carboxyfluoresceïne diacetaat succinimidylester (CFSE) kan worden gebruikt om eenvoudig en snel het etiket een cel populatie van belang zijn voor in vivo onderzoek. Deze etikettering is klassiek is gebruikt om de proliferatie en migratie te bestuderen. In de hier gepresenteerde methode, hebben we verkort de tijdlijn na adoptieve overdracht om te kijken naar overleving en doden van epitoop specifieke CFSE gelabelde doelcellen 4-6. Het niveau van specifieke doden van een CD8 + T-cel-kloon kan duiden op de kwaliteit van de reactie, aangezien hun hoeveelheid kan misleidend zijn. Specifieke CD8 + T-cellen kunnen functioneel uitgeput raken na verloop van tijd met een daling van de cytokine productie en het doden van 7,8. Ook kunnen bepaalde CD8 + T cel klonen niet zo goed als anderen te doden met verschillende TCR specifieke 9. Voor een effectieve cel gemedieerde immuniteit (CMI), moet antigenen worden geïdentificeerd die produceren niet alleen een voldoende aantal te reageren T-cellen, maar ook functioneel robuuste reageren T-cellen. Hier beoordelen we het percentage celspecifieke moord op twee peptide specifieke T-cel klonen in BALB / c muizen.

Watch this Scientific Journal Video about Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells at JoVE.com

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Veel infecties roepen een sterke CTL-respons, maar af en toe, de hoeveelheid cellen die reageren niet correleren met de controle van de ziekteverwekker<sup&gt; 1</sup&gt;. Een maat van CTL kwaliteit is hun vermogen om specifiek te doden<sup&gt; 2</sup&gt;. CFSE labeling van target cellen kunnen worden gebruikt om deze CTL-respons kwaliteit te onderzoeken<em&gt; In vivo</em<sup&gt; 3,4</sup&gt;.

Antigen Specifieke In Vivo Killing Assay met behulp van CFSE Labeled doelcellen

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

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