Name: extraction of tissue antigens for functional assays
Uploaded: 2012-09-10T13:00:00
Description: Watch this Scientific Journal Video about Extraction of Tissue Antigens for Functional Assays at JoVE.com

Dette arbejde er støttet af tilskud fra Australian National Sundhed og Medical Research Council (NHMRC # 559.007) og den Juvenile Diabetes Research Foundation (JDRF 4-2006-1025) og den operationelle infrastruktur under Den victorianske regering. Vi takker medlemmerne af Tom Mandel Islet Transplantation Program Islet Isolation Team for at sikre de menneskelige væv. Humane væv blev opsamlet og anvendt med lokal etisk godkendelse (St. Vincents Hospital HREC-A 011/04 og St. Vincent Health HREC-A 135/08).

1. Fremstilling miltvæv <ol><li> Note-al menneskelig materiale skal behandles som potentielt smittefarlige og alle procedurer skal udføres i et Klasse II Laminar Flow kabinet. Ved hjælp af steril saks og pincet, fjerne fedt og fibrøst væv fra milt sektioner (~ 1-2 cm i størrelse) og trimme så meget af den ydre kapsel materiale som muligt. </li><li> Skær et lille stykke (1-2 cm 3) i miltvæv og placere hvert stykke i et sterilt 50 ml Falcon-rør. </li><li> Snap-fryse stykker af væv ved nedsænkning ...

<ol>
	<li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+to+measuring+human+islet-antigen+specific+T+cell+function+in+type+1+diabetes.">Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.</a> Clin. Exp. Immunol. 162, 197-209 (2010).</li><li>Beckhove, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+T+cell-based+identification+of+human+tumor+tissue+antigens+by+automated+two-dimensional+protein+fractionation.">Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.</a> J. Clin. Invest. 120, 2230-2242 (2010).</li><li>Mannering, S. I., Brodnicki, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+CD4++T-cell+specificity+and+function+in+Type+1+diabetes.">Recent insights into CD4+ T-cell specificity and function in Type 1 diabetes.</a> Expert Review of Clinical Immunology. 3, 557-564 (2007).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+A-chain+of+insulin+is+a+hot-spot+for+CD4++T+cell+epitopes+in+human+type+1+diabetes.">The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.</a> Clin. Exp. Immunol. 156, 226-231 (2009).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+A-chain+epitope+recognized+by+human+T+cells+is+posttranslationally+modified.">The insulin A-chain epitope recognized by human T cells is posttranslationally modified.</a> J. Exp. Med. 202, 1191-1197 (2005).</li><li>Moon, H. C., Joffe, M., Thomas, H. E., Kay, T. W. H., Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+extracting+tissue+proteins+for+use+in+lymphocyte+function+assays.">A method for extracting tissue proteins for use in lymphocyte function assays.</a> Journal of Immunological Methods. 359, 56-60 (2010).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J. Immunol. Methods. 283, 173-183 (2003).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+Cell+Proliferation+in+Response+to+GAD+and+Proinsulin+in+Healthy,+Pre-diabetic,+and+Diabetic+Donors.">CD4+ T Cell Proliferation in Response to GAD and Proinsulin in Healthy, Pre-diabetic, and Diabetic Donors.</a> Ann. N.Y. Acad. Sci. 1037, 16-21 (2004).</li><li>Mannering, S. I., Purcell, A. W., Honeyman, M. C., McCluskey, J., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T-cells+recognise+N-terminally+Fmoc-modified+peptide.">Human T-cells recognise N-terminally Fmoc-modified peptide.</a> Vaccine. 21, 3638-3646 (2003).</li><li>Peakman, M. <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+preparations+of+GAD65,+proinsulin,+and+the+islet+tyrosine+phosphatase+IA-2+for+use+in+detection+of+autoreactive+T-cells+in+type+1+diabetes:+report+of+phase+II+of+the+Second+International+Immunology+of+Diabetes+Society+Workshop+for+Standardization+of+T-cell+assays+in+type+1+diabetes.">Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.</a> Diabetes. 50, 1749-1754 (2001).</li><li>Honey, K., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+cysteine+proteases+regulate+antigen+presentation.">Lysosomal cysteine proteases regulate antigen presentation.</a> Nat. Rev. Immunol. 3, 472-482 (2003).</li></ol>

Denne protokol blev udviklet, fordi vi ønskede at generere et uddrag fra humant væv, der var fri for giftige kemikalier såsom vaskemidler. Specifikt har vi anvendt det til fremstilling af ekstrakter af humane væv, der kan anvendes i assays af human immunfunktion in vitro. Ekstrakter fremstillet under anvendelse af denne protokol kan også rekonstitueres i en buffer og anvendes til mange biokemiske analyser, såsom Western blotting eller væskekromatografi. Dette gør denne teknik anvendes på mange efterfø...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> 5 ml 12 x 75 mm sterile polystyrenrør </td><td> BD Falcon </td><td> 352.054 </td><td></td></tr><tr><td> Caps til rør polystyrenrør (ovenfor) </td><td> BD Falcon </td><td> 352.032 </td><td></td></tr><tr><td> 50ml sterile rør </td><td> Becton Dickinson </td><td> 352.070 </td><td></td></tr><tr><td> Acetonitril </td><td> Mallinckradt Chemicals </td><td> 2856-10 </td><td></td></tr><tr><td> Butan-1-ol </td><td> Sigma Aldrich </td><td> 537993-IL </td><td></td></tr><tr><td> Homogenizer: PRO200 </td><td> Bio-strategien </td><td> 01-01200 </td><td> 10 x 115 mm savtakket generator </td></tr><tr><td> Frysetørringsapparat </td><td></td><td></td><td> Virtis, Benchtop 4K </td></tr><tr><td> Steril nål 18-20 gauge </td><td> Becton Dickinson </td><td> REF 302.032 </td><td></td></tr><tr><td> CMRL-1066 medium </td><td> Sigma </td><td> C0422 </td><td></td></tr><tr><td> PBS </td><td> Sigma </td><td> D8537 </td><td></td></tr></tbody></table> Tabel 1. Specifikke reagenser og udstyr.

extraction of tissue antigens for functional assays

Mange af de antigen-mål for adaptive immunrespons, genkendes af B-og T-celler, er ikke blevet defineret 1. Dette gælder især i autoimmune sygdomme og cancer 2. Vores mål er at undersøge de antigener genkendes af humane T-celler i den autoimmune sygdom type 1 diabetes 1,3,4,5. At analysere humane T-cellereaktioner mod vævet, hvor de antigener, der genkendes af T-celler identificeres ikke udviklede vi en metode til at udvinde proteinantigener fra humant væv i et format der er kompatibelt med funktionelle assays 6. Tidligere T-celle-reaktioner på urensede vævsekstrakter kunne ikke måles, fordi ekstraktionsmetoder opnåelse af et lysat, som indeholdt detergenter, der var toksiske for humane perifere mononukleære blodceller. Her beskriver vi en protokol til ekstraktion af proteiner fra humane væv i et format, der ikke er toksisk for humane T-celler. Vævet homogeniseres i en blanding af butan-1-ol, acetonitril og watis (BAW). Proteinkoncentrationen i vævsekstrakt måles, og en kendt masse af protein alikvoter i rør. Efter ekstraktion, er de organiske opløsningsmidler fjernet ved lyofilisering. Lyofiliserede vævsekstrakter kan lagres indtil brug. Til anvendelse i assays af immunfunktion, en suspension af immunceller, i egnede dyrkningsmedier kan tilsættes direkte til den frysetørrede ekstrakt. Cytokinproduktion og proliferation af PBMC, som svar på ekstrakter fremstillet ved denne metode, kunne let måles. Derfor er vores fremgangsmåde tillader hurtig fremstilling af humant væv lysater, der kan anvendes som en kilde til antigener i analysen af ​​T-cellereaktioner. Vi foreslår, at denne metode vil lette analysen af ​​adaptive immunresponser på væv i transplantation, cancer og autoimmunitet.

Watch this Scientific Journal Video about Extraction of Tissue Antigens for Functional Assays at JoVE.com

Extraction of Tissue Antigens for Functional Assays

En enkel protokol til fremstilling af ekstrakter af humane væv til anvendelse som en kilde til antigener i funktionelle T-celleassays er beskrevet. Denne fremgangsmåde tillader T-celle-reaktioner på vævs-afledte antigener, der skal måles<em&gt; In vitro</em&gt;.

Ekstraktion af Tissue Antigens for Funktionelle analyser

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

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