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

We willen graag Marcelo Toro erkennen voor de hulp bij flowcytometrie. Deze studie werd ondersteund door de Zweedse Hart-en Long Foundation, EU-Commissie KP7 &quot;CardioCell&quot;, Zweedse Raad voor Onderzoek, AFA verzekeringen en ALF. OB werd ondersteund door de Deutsche Forschungsgemeinschaft.

1. Isolatie van de Cardiac Kernen <ol><li> Coat ultracentrifuge buizen (Beckman centrifugebuizen # 363664) met 10 ml van 1% BSA / PBS coating oplossing. Cap de buizen en laten roteren gedurende 30 min in een buis rotator. Verwijder de coatingoplossing en laat de centrifugebuis drogen (een buis per muizenhart is voor de analyse van de muis hart, afwisselend tot 5 muizen hart of 1 g hartweefsel van een andere soort (bijv. humane) kunnen worden verwerkt in een buis). </li><li> Alle volgende stappen moeten worden uitgevoe...

<ol>
	<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>

Nauwkeurige identificatie van cardiomyocyt kernen is van cruciaal belang voor de analyse van regeneratieve processen in het myocard 2,3. Conventionele technieken voor cardiomyocyten van sel isoleren zijn hoofdzakelijk gebaseerd op enzymatische afbraak van de extracellulaire matrix en de daaropvolgende zuivering van interstitiële cellen door centrifugeren lage snelheid. Verdere zuivering van levende cardiomyocyten van embryonale stamcellen (SER) kan worden uitgevoerd door immunokleuring met markers zoals Sirp...

<table border="1"><tbody><tr><td> 1. Lysisbuffer </td></tr><tr><td> Naam van het serum </td></tr><tr><td> 0,32 M sucrose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl2 </td></tr><tr><td> 5 mM magnesiumacetaat </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. Sucrose buffer </td></tr><tr><td> Naam van het serum </td></tr><tr><td> 2.1 M sucrose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM magnesiumacetaat </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Kernen opslagbuffer (NSB plus) </td></tr><tr&gt; <td> Naam van het serum </td></tr><tr><td> 0,44 M sucrose </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 spermine </td></tr></tbody></table><table border="1"><tbody><tr><td> Reagentia en apparatuur </td><td> Vennootschap </td></tr><tr><td> Isotype konijnen-IgG-CHIP Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> Konijn anti-PCM-1 antilichaam, # HPA023374 </td><td> Atlas Antilichamen </td></tr><tr><td> Donkey sec. antilichaam, anti-konijn Alexa Fluor 488, # A-21206 of een gelijkwaardige sec. fluorescentie-antilichaammethode </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> cell filters 30 pm, 70 pm en 100 pm </td><td> BD Biosciences </td></tr><tr><td> Glas douncer (40 ml) enstamper &quot;L&quot; </td><td> VWR (Wheaton Industries Inc) </td></tr><tr><td> T-25 Ultra-Turrax Homogenizer </td><td> IKA Duitsland </td></tr><tr><td> Dispergeren hulpmiddel S25 N-18 G </td><td> IKA Duitsland </td></tr><tr><td> Beckman Avanti Centrifuge </td><td> Beckman Coulter </td></tr><tr><td> Falcon Buizen 15 ml en 50 ml </td><td> VWR </td></tr><tr><td> Beckman Centrifugebuizen # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 gratis swingende rotor </td><td> Beckman Coulter </td></tr><tr><td> Instroom cytometer </td><td> Beckman Coulter </td></tr><tr><td> Tube Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identificatie van cardiomyocyten kernen is een uitdaging in het weefsel secties als de meeste strategieën vertrouwen op cytoplasmatische marker-eiwitten 1. Zeldzame gebeurtenissen in hartspiercellen zoals de proliferatie en apoptose vereisen een nauwkeurige identificatie van de cardiale myocyten kernen om celvernieuwing in de homeostase en in pathologische voorwaarden 2 te analyseren. Hier bieden we een methode om cardiomyocyt kernen te isoleren van post mortem weefsel door de dichtheid sedimentatie en immunokleuring met antilichamen tegen pericentriolar materiaal 1 (PCM-1) en de daarop volgende flowcytometrie sorteren. Deze strategie zorgt voor een hoge doorvoer analyse en isolatie met het voordeel van het werken even goed op verse en bevroren weefsel archiefmateriaal. Dit maakt het mogelijk te onderzoeken materiaal reeds verzamelde biobanken. Deze techniek is en getest in een groot aantal soorten en geschikt voor diverse toepassingen later zoals koolstof-14 datering 3 cel-cykel analyse 4, visualisatie van thymidine-analogen (bv. BrdU en IDU) 4, transcriptoom en epigenetische analyse.

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

Cardiale kernen worden geïsoleerd via dichtheid sedimentatie en immunolabeled met antilichamen tegen pericentriolar materiaal 1 (PCM-1) te identificeren en cardiomyocyten kernen sorteren op flowcytometrie.

Isolatie van cardiomyocyten Kernen van Post-mortem weefsel

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

Research

JoVE Journal

Medicine

Isolation of Human Islets from Partially Pancreatectomized Patients

Isolatie van de Mens Eilandjes van Gedeeltelijk Pancreatectomized Patiënten

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Isolatie van kanker stamcellen uit menselijke stalen van prostaatkanker

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Isolatie en profilering van MicroRNA-bevattende exosomes van Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

Bemonsteringsstrategie en verwerking van Biobank weefselmonsters van varkens biomedische modellen

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Isolation of Atrial Myocytes from Adult Mice

Isolatie van atriale Myocyten van volwassen muizen

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Elektromechanische beoordeling van optogenetisch gemoduleerde cardiomyocyteactiviteit

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Model van ischemische hartziekten en video-gebaseerde vergelijking van Cardiomyocyte Contractie met behulp van hiPSC-afgeleide Cardiomyocyten

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Isolatie van levensvatbare vetcellen en stromale vasculaire fractie uit menselijk visceraal vetweefsel geschikt voor RNA-analyse en macrofaagfenotypering

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Voorbereiding van een niet-cardiomyocytencelsuspensie voor single-cell RNA-sequencing van een post-myocardinfarct volwassen muizenhart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...