Name: analysis of pulmonary dendritic cell maturation and migration during allergic airway inflammation
Uploaded: 2012-07-23T12:00:00
Description: Watch this Scientific Journal Video about Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation at JoVE.com

Dette arbejde blev finansieret af CIHR og CF Canada tilskud til Dr. Jim Hu og en ph.d. studentship tildelt Rahul Kushwah af CF Canada.

1. Sensibilisering af mus med ovalbumin <ol><li> Fremstille en opløsning af OVA (kvalitet V, Sigma, MO) i sterilt PBS til en koncentration på 1 mg / ml (opløsning kan opbevares ved -80 ° C). </li><li> For at forberede OVA-Alun blanding tage alun i et rør, og der tilsættes OVA opløsning på en dråbevis måde mens prøveglasset omrystes i et forhold på 1:1. Blandingen omrøres i 30 minutter og anvende det samme efter blanding. </li><li> Anvendelse af en 1 ml sprøjte, injiceres 0,2 ml af blandingen i mus perito...

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	<li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+dendritic+cells+in+the+induction+of+regulatory+T+cells.">Role of dendritic cells in the induction of regulatory T cells.</a> Cell Biosci. 20, (2011).</li><li>Kushwah, R., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+of+dendritic+cell+subsets+and+their+function+in+the+host+immune+system.">Complexity of dendritic cell subsets and their function in the host immune system.</a> Immunology. 133, 409-419 (2011).</li><li>Lambrecht, B. N., Hammad, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+our+breath+away:+dendritic+cells+in+the+pathogenesis+of+asthma.">Taking our breath away: dendritic cells in the pathogenesis of asthma.</a> Nat Rev Immunol. 3, 994-1003 (2003).</li><li>Hammad, H., Lambrecht, B. N. <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+and+epithelial+cells:+linking+innate+and+adaptive+immunity+in+asthma.">Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma.</a> Nat. Rev. Immunol. 8, 193-204 (2008).</li><li>Lloyd, C. M., Murdoch, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerizing+allergic+responses+in+the+lung.">Tolerizing allergic responses in the lung.</a> Mucosal. Immunol. 3, 334-344 (2010).</li><li>Herz, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+murine+animal+models+to+study+the+development+of+allergic+bronchial+asthma.">The relevance of murine animal models to study the development of allergic bronchial asthma.</a> Immunol. Cell Biol. 74, 209-217 (1038).</li><li>Herz, U., Lumpp, U., Daser, A., Gelfand, E. W., Renz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+animal+models+to+study+the+central+role+of+T+cells+in+immediate-type+hypersensitivity+responses.">Murine animal models to study the central role of T cells in immediate-type hypersensitivity responses.</a> Adv. Exp. Med. Biol. 409, 25-32 (1996).</li><li>Nakae, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF+can+contribute+to+multiple+features+of+ovalbumin-induced+allergic+inflammation+of+the+airways+in+mice.">TNF can contribute to multiple features of ovalbumin-induced allergic inflammation of the airways in mice.</a> J. Allergy Clin. Immunol. 119, 680-686 (2007).</li><li>Guth, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+environment+determines+unique+phenotype+of+alveolar+macrophages.">Lung environment determines unique phenotype of alveolar macrophages.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 296, L936-L946 (2009).</li><li>Kushwah, R., Cao, H., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pulmonary+T+cell+response+to+helper-dependent+adenoviral+vectors+following+intranasal+delivery.">Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery.</a> J. Immunol. 180, 4098-4108 (2008).</li><li>Kushwah, R., Oliver, J. R., Zhang, J., Siminovitch, K. A., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+dendritic+cells+induce+tolerance+in+mice+through+suppression+of+dendritic+cell+maturation+and+induction+of+antigen-specific+regulatory+T+cells.">Apoptotic dendritic cells induce tolerance in mice through suppression of dendritic cell maturation and induction of antigen-specific regulatory T cells.</a> J. Immunol. 183, 7104-7118 (2009).</li></ol>

Metoden præsenteres her tilbyder en flowcytometri tilgang til analyse pulmonale DCS, baseret på levering af OVA-FITC for luftvejene udfordring. Dette muliggør selektiv kontrol af pulmonære DC&#39;er, som optager OVA-FITC og derfor DC populationer, der overvåges, er effektivt dem, der deltager i luftvejene immunrespons i løbet af OVA-induceret allergisk luftvejsinflammation. I kontrolmus, i fravær af allergisk luftvejsinflammation antallet af migrerende DCS (OVA-FITC + DC&#39;er) er identificeret i MLN indikerer b...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Ovalbumin </td><td> Sigma-Aldrich </td><td> A5503 </td></tr><tr><td> Alum </td><td> Thermo Scientific </td><td> 77161 </td></tr><tr><td> OVA-FITC </td><td> Sigma-Aldrich </td><td> A9771 </td></tr><tr><td> Collagenase D </td><td> Roche </td><td> 11088858001 </td></tr><tr><td> Antistoffer </td><td> e-Bioscience </td><td></td></tr></tbody></table>

analysis of pulmonary dendritic cell maturation and migration during allergic airway inflammation

Dendritiske celler (DC&#39;er) er de hovedaktører involveret i initiering af adaptive immunreaktion ved at aktivere antigenspecifikke T-celler. DC&#39;er er til stede i perifere væv i steady state, men som respons på antigen stimulering, DC&#39;er optage antigenet og hurtigt migrere til de drænende lymfeknuder, hvor de initierer T celle-respons mod antigenet 1,2. Derudover udviklingslande også spille en vigtig rolle i at indlede autoimmun samt allergisk immunrespons 3. Udviklingslandene spiller en afgørende rolle både i initiering af immunrespons og induktion af tolerance i indstilling af lunge miljøet 4. Lunge miljø er hovedsagelig tolerogen grund af udsættelse for lang række miljømæssige antigener 5. Men i nogle individer der er en pause i tolerance, hvilket fører til induktion af allergi og astma. I denne undersøgelse beskriver vi en strategi, der kan anvendes til at overvåge luftvejene DC modning og MIGration som respons på antigenet anvendt til sensibilisering. Måling af luftvejstryk DC modning og migration tillader vurdering af kinetikken af ​​immunrespons i luftvejene allergisk inflammation og også hjælper til at forstå omfanget af de efterfølgende immunrespons sammen med de underliggende mekanismer. Vor strategi er baseret på anvendelsen af ​​ovalbumin som et sensibiliserende middel. Ovalbumin-induceret allergisk astma er en almindeligt anvendt model til at reproducere luftvejene eosinofili, pulmonal inflammation og forhøjede IgE-niveauer blev fundet under astma 6,7. Efter sensibilisering, mus udfordret ved intranasal levering af FITC-mærket ovalbumin, som tillader specifik mærkning af luftvejs DC&#39;er hvor optagelse ovalbumin. Dernæst, ved hjælp af flere DC specifikke markører, kan vi vurdere modningen af ​​disse udviklingslandene og kan også vurdere deres migration til de drænende lymfeknuder ved at ansætte flowcytometri.

Watch this Scientific Journal Video about Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation at JoVE.com

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Vi beskriver en strategi til at overvåge modning og migrering af pulmonære dendritiske celler som respons på ovalbumin i opstillingen af ​​ovalbumin-induceret allergisk luftvejsinflammation. Denne strategi kan modificeres til at vurdere migrering af pulmonale dendritiske celler i indstillinger for infektion.

Analyse af Pulmonal dendritiske celle modning og migration under Allergisk luftvejene

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

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