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

Nos gustaría agradecer Marcelo Toro para la asistencia con la citometría de flujo. Este estudio fue apoyado por el sueco de corazón y pulmón Fundación, la Comisión Europea el 7 º PM &quot;CardioCell&quot;, Consejo Sueco de Investigación, seguros AFA y ALF. OB fue apoyado por la Deutsche Forschungsgemeinschaft.

1. El aislamiento de los Núcleos cardiaca <ol><li> Tubos Coat ultracentrífuga Beckman (Tubos de centrífuga # 363664) con 10 ml de solución de revestimiento 1% de BSA / PBS. Tape los tubos y dejar que gire durante 30 minutos en un rotor de tubo. Eliminar la solución de revestimiento y dejar que la centrífuga tubos de aire seco (un tubo por el corazón del ratón se requiere para el análisis de los corazones de ratón individuales, alternativamente hasta 5 corazones de ratón o 1 g de tejido cardíaco de una espe...

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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>

La identificación precisa de los núcleos de cardiomiocitos es crucial para el análisis de los procesos regenerativos en el miocardio 2,3. Las técnicas convencionales para aislar cardiomiocitos a partir de tejido fresco se basan principalmente en la digestión enzimática de proteínas de matriz extracelular y la posterior purificación de las células intersticiales por centrifugación a baja velocidad. La purificación adicional de cardiomiocitos vivos a partir de células madre embrionarias (CES) puede ...

<table border="1"><tbody><tr><td> 1. Tampón de lisis </td></tr><tr><td> Nombre del reactivo </td></tr><tr><td> 0,32 M de sacarosa </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de CaCl 2 </td></tr><tr><td> 5 mM de acetato de magnesio </td></tr><tr><td> 2,0 mM de EDTA </td></tr><tr><td> 0,5 mM EGTA </td></tr><tr><td> 1 mM de DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Sacarosa tampón </td></tr><tr><td> Nombre del reactivo </td></tr><tr><td> 2,1 M de sacarosa </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM de acetato de magnesio </td></tr><tr><td> 1 mM de DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Los núcleos buffer de almacenamiento (NSB más) </td></tr><tr&gt; <td> Nombre del reactivo </td></tr><tr><td> 0,44 M de sacarosa </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM de KCl </td></tr><tr><td> 10 mM MgCl 2 </td></tr><tr><td> 1,5 mM de espermina </td></tr></tbody></table><table border="1"><tbody><tr><td> Reactivos y Equipos </td><td> Empresa </td></tr><tr><td> Isotipo IgG de conejo-chip de grado, # ab37415 </td><td> Abcam </td></tr><tr><td> De conejo anti-PCM-1 anticuerpo, # HPA023374 </td><td> Atlas anticuerpos </td></tr><tr><td> Donkey seg. anticuerpos, anti-conejo Alexa 488 Fluor, # A-21206 o equivalente seg. anticuerpos fluorescentes </td><td> Tecnologías de la Vida </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> coladores de células de 30 m, micras y 70 micras 100 </td><td> BD Biosciences </td></tr><tr><td> Vidrio douncer (40 ml) y sela mano del mortero &quot;L&quot; </td><td> VWR (Wheaton Industries Inc.) </td></tr><tr><td> T-25 Ultra-Turrax </td><td> IKA Alemania </td></tr><tr><td> La dispersión de herramientas S25 N-18 G </td><td> IKA Alemania </td></tr><tr><td> Beckman Avanti Centrífuga </td><td> Beckman Coulter </td></tr><tr><td> Tubos Falcon de 15 ml y 50 ml </td><td> VWR </td></tr><tr><td> Tubos de centrífuga Beckman # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 rotor de giro libre </td><td> Beckman Coulter </td></tr><tr><td> Afluencia citómetro </td><td> Beckman Coulter </td></tr><tr><td> Tubo de los rotadores </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identificación de los núcleos de cardiomiocitos ha sido un desafío en secciones de tejido, como la mayoría de las estrategias se basan sólo en proteínas marcadoras frente al citoplasma de 1. Los eventos raros en los miocitos cardíacos, como la proliferación y la apoptosis requiere una identificación precisa de los núcleos de los miocitos cardíacos para analizar la renovación celular en la homeostasis y en condiciones patológicas 2. En este caso, se proporciona un método para aislar núcleos de cardiomiocitos de post mortem del tejido por la sedimentación y la densidad de inmunomarcación con anticuerpos contra el material pericentriolar 1 (PCM-1) y la clasificación posterior de citometría de flujo. Esta estrategia permite un análisis de alto rendimiento y el aislamiento con la ventaja de trabajar igualmente bien en tejido fresco y material de archivo congelado. Esto hace posible el estudio de material ya reunido en biobancos. Esta técnica es aplicable y probado en una amplia gama de especies y adecuado para múltiples aplicaciones posteriores tales como el carbono-14 contactos 3, célula-CYculo 4 de análisis, la visualización de los análogos de timidina (por ejemplo, BrdU y UDI) 4, el análisis de transcriptoma y epigenéticos.

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

Núcleos están aislados cardíaca a través de la sedimentación y la densidad de immunolabeled con anticuerpos contra el material pericentriolar 1 (PCM-1) para identificar y clasificar los núcleos de cardiomiocitos mediante citometría de flujo.

El aislamiento de núcleos de cardiomiocitos a partir de tejidos post-mortem

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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