Name: isolation of cardiomyocyte nuclei from post-mortem tissue
Uploaded: 2012-07-10T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Vi gillar att erkänna Marcelo Toro för hjälp med flödescytometri. Denna studie stöddes av svenska Hjärt-och Lungfonden, EU-kommissionen FP7 &quot;CardioCell&quot;, Svenska Vetenskapsrådet, AFA försäkringar och ALF. OB stöddes av Deutsche Forschungsgemeinschaft.

1. Isolering av Cardiac Kärnor <ol><li> Belägga ultracentrifugrör (Beckman Centrifugrör # 363664) med 10 ml av 1% BSA / PBS beläggningslösning. Förslut rören och låt dem rotera under 30 min i ett rör rotator. Avlägsna beläggningslösningen och låta centrifugrör lufttorka (ett rör per mushjärta erfordras för analys av enstaka mus hjärtan, alternativt upp till 5 hjärtan från mus eller 1 g av hjärtvävnad från en annan art (t.ex. människa) kan bearbetas i ett rör). </li><li> Alla följande åtgär...

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	<li>Ang, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+conventional+approaches+to+identify+myocyte+nuclei+in+histologic+sections+of+the+heart.">Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart.</a> American journal of physiology. Cell physiology. , 298-1603 (2010).</li><li>Bergmann, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardiomyocyte+nuclei+and+assessment+of+ploidy+for+the+analysis+of+cell+turnover.">Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.</a> Experimental cell research. 327, 188-194 (2011).</li><li>Bergmann, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Cardiomyocyte+Renewal+in+Humans.">Evidence for Cardiomyocyte Renewal in Humans.</a> Science. 324, 98-102 (1126).</li><li>Walsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+cell+cycle+control+and+growth+estimation.">Cardiomyocyte cell cycle control and growth estimation.</a> Cardiovascular Research. , 1-31 (2010).</li><li>Spalding, K., Bhardwaj, R. D., Buchholz, B., Druid, H., Frisén, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Adler, C. P., Friedburg, H., Herget, G. W., Neuburger, M., Schwalb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+of+cardiomyocyte+DNA+content,+ploidy+level+and+nuclear+number+in+mammalian+hearts.">Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts.</a> Virchows Arch. 429, 159-164 (1996).</li><li>Herget, G. W., Neuburger, M., Plagwitz, R., Adler, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content,+ploidy+level+and+number+of+nuclei+in+the+human+heart+after+myocardial+infarction.">DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction.</a> Cardiovascular Research. 36, 45-51 (1997).</li><li>Adler, C. P., Friedburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+DNA+content.+ploidy+level+and+cell+number+in+geriatric+hearts:+postmortem+examinations+of+human+myocardium+in+old+age.">Myocardial DNA content. ploidy level and cell number in geriatric hearts: postmortem examinations of human myocardium in old age.</a> Mol. Cell Cardiol. 18, 3953-39 (1986).</li><li>Dubois, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRPA+is+a+specific+cell-surface+marker+for+isolating+cardiomyocytes+derived+from+human+pluripotent+stem+cells.">SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.</a> Nature. 29, 1011-1018 (2011).</li><li>Hattori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nongenetic+method+for+purifying+stem+cell-derived+cardiomyocytes.">Nongenetic method for purifying stem cell-derived cardiomyocytes.</a> Nature Methods. 7, 61-66 (2010).</li><li>Fransioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+c-kit-Positive+Cell+Response+to+Pathological+Challenge+in+the+Myocardium.">Evolution of the c-kit-Positive Cell Response to Pathological Challenge in the Myocardium.</a> Stem Cells. 26, 1315-1324 (2008).</li><li>Elliott, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKX2-5(eGFP/w)+hESCs+for+isolation+of+human+cardiac+progenitors+and+cardiomyocytes.">NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.</a> Nature Methods. 8, 1037-1040 (2011).</li><li>Laflamme, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Cardiomyocyte+Repopulation+by+Extracardiac+Progenitors+in+Transplanted+Human+Hearts.">Evidence for Cardiomyocyte Repopulation by Extracardiac Progenitors in Transplanted Human Hearts.</a> Circulation Research. 90, 634-640 (2002).</li><li>Srsen, V., Fant, X., Heald, R., Rabouille, C., Merdes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+proteins+form+an+insoluble+perinuclear+matrix+during+muscle+cell+differentiation.">Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation.</a> BMC cell biology. 10, 28 (2009).</li><li>Spoelgen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometry-based+technique+to+measure+adult+neurogenesis+in+the+brain.">A novel flow cytometry-based technique to measure adult neurogenesis in the brain.</a> Journal of neurochemistry. 119, 165-175 (2011).</li><li>Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+DNA+synthesis+and+binucleation+during+murine+development.">Cardiomyocyte DNA synthesis and binucleation during murine development.</a> The American journal of physiology. 271, H2183-H2189 (1996).</li><li>Olivetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+cardiac+hypertrophy+and+ischemic+cardiomyopathy+do+not+affect+the+proportion+of+mononucleated+and+multinucleated+myocytes+in+the+human+heart.">Aging, cardiac hypertrophy and ischemic cardiomyopathy do not affect the proportion of mononucleated and multinucleated myocytes in the human heart.</a> J Mol Cell Cardiol. 28, 1463-1477 (1996).</li><li>Okada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+sorting+of+neuronal+and+glial+nuclei+from+central+nervous+system+tissue.">Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue.</a> Journal of cellular physiology. 226, 552-558 (2011).</li><li>Matevossian, A., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Nuclei+Isolation+from+Human+Postmortem+Brain+Tissue.">Neuronal Nuclei Isolation from Human Postmortem Brain Tissue.</a> J. Vis. Exp. (20), e914 (2008).</li></ol>

Exakt identifiering av kardiomyocetisk kärnor är avgörande för analys av regenerativa processer i hjärtmuskeln 2,3. Konventionella tekniker för att isolera kardiomyocyter från färsk vävnad är huvudsakligen baserade på enzymatisk digestion av extracellulära matrisproteiner och efterföljande rening av interstitiella celler genom centrifugering vid låg hastighet. Ytterligare rening av levande hjärtmuskelceller från embryonala stamceller (ESC) kan utföras av immunomärkning med ytmarkörer som Si...

<table border="1"><tbody><tr><td> 1. Lysbuffert </td></tr><tr><td> Namnet på reagens </td></tr><tr><td> 0,32 M sackaros </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl2 </td></tr><tr><td> 5 mM magnesiumacetat </td></tr><tr><td> 2,0 mM EDTA </td></tr><tr><td> 0,5 mM EGTA </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Sackarosbuffert </td></tr><tr><td> Namnet på reagens </td></tr><tr><td> 2,1 M sackaros </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM magnesiumacetat </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Kärnor lagringsbuffert (NSB plus) </td></tr><tr&gt; <td> Namnet på reagens </td></tr><tr><td> 0,44 M sackaros </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM KCl </td></tr><tr><td> 10 mM MgCl2 </td></tr><tr><td> 1,5 mM spermin </td></tr></tbody></table><table border="1"><tbody><tr><td> Reagens och utrustning </td><td> Företaget </td></tr><tr><td> Isotyp IgG-CHIP Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> Kanin-anti-PCM-1-antikropp, # HPA023374 </td><td> Atlas Antibodies </td></tr><tr><td> Åsna sek. antikropp, anti-kanin Alexa Fluor 488, # A-21.206 eller motsvarande sek. fluorescerande antikroppar </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> cell silar 30 pm, 70 pm och 100 pm </td><td> BD Biosciences </td></tr><tr><td> Glas douncer (40 ml) ochpistill &quot;L&quot; </td><td> VWR (Wheaton Industries Inc.) </td></tr><tr><td> T-25 Ultra-Turrax-homogenisator </td><td> IKA Tyskland </td></tr><tr><td> Dispergermedel verktyg S25 N-18 G </td><td> IKA Tyskland </td></tr><tr><td> Beckman Avanti Centrifugera </td><td> Beckman Coulter </td></tr><tr><td> Falcon-rör 15 ml och 50 ml </td><td> VWR </td></tr><tr><td> Beckman Centrifugrör # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 fri svängande rötor </td><td> Beckman Coulter </td></tr><tr><td> Inflöde cytometer </td><td> Beckman Coulter </td></tr><tr><td> Röret Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identifiering av kardiomyocetisk kärnor har varit en utmaning i vävnadssnitt som de flesta strategierna förlita sig enbart på cytoplasmiska markörproteiner 1. Sällsynta händelser i hjärtmuskelceller som proliferation och apoptos kräver en noggrann kartläggning av hjärtmuskelceller kärnor för att analysera cellulära förnyelse i homeostas och patologiska tillstånd 2. Här ger vi en metod för att isolera kardiomyocetisk kärnor från post mortem vävnad genom täthet sedimentering och immunomärkning med antikroppar mot pericentriolar material 1 (PCM-1) och efterföljande flödescytometri sortering. Denna strategi gör det möjligt för en hög genomströmning analys och isolering med fördelen att arbeta lika bra på färsk vävnad och fryst arkivmaterial. Detta gör det möjligt att studera material som redan samlats in i biobanker. Denna teknik är tillämplig och testas i ett brett spektrum av arter och passar för flera nedströms applikationer såsom kol-14 datering 3, cell-cyCLE Analys 4, visualisering av tymidinanaloger (t.ex. BrdU och IDU) 4, transkriptom och epigenetiska analys.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Hj Kärnor isoleras via densitet sedimentering och immunolabeled med antikroppar mot pericentriolar materialet 1 (PCM-1) för att identifiera och sortera kardiomyocyter kärnor med flödescytometri.

Isolering av kardiomyocetisk Kärnor från Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

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