Name: visualization of cell cycle variations and determination of nucleation in postnatal cardiomyocytes
Uploaded: 2017-02-24T14:00:00
Description: Watch this Scientific Journal Video about Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes at JoVE.com

We thank S. Gr&#252;nberg (Bonn, Germany) and P. Freitag (Bonn, Germany) for their technical assistance.

Alle Verfahren dieses Protokolls Tiere waren in Einklang mit den ethischen Normen der Universität Bonn und mit den Leitlinien der Richtlinie 2010/63 / EU des Europäischen Parlaments über den Schutz von Tieren für wissenschaftliche Zwecke erfüllt. 1. In - vitro - Visualisierung von Zellzyklusaktivität in postnatale Kardiomyozyten <ol><li> Die postnatale Kardiomyozyten Dissoziation <ol><li> Pre-experimentelle Präparate <ol><li> Bereiten Kulturmedien (IMDM, 1% Penicil...

<ol>
	<li>Bergmann, O., et al. <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 (5923), 98-102 (2009).</li><li>Raulf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+systems+for+unequivocal+identification+of+cardiac+myocyte+nuclei+and+analysis+of+cardiomyocyte+cell+cycle+status.">Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status.</a> Basic Res.Cardiol. 110 (3), 33 (2015).</li><li>Field, C. M., Alberts, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin,+a+contractile+ring+protein+that+cycles+from+the+nucleus+to+the+cell+cortex.">Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.</a> J.Cell Biol. 131 (1), 165-178 (1995).</li><li>Hesse, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+cell+division+using+high-resolution+imaging+of+M-phase+of+the+cell+cycle.">Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle.</a> Nat.Commun. 3, 1076 (2012).</li><li>Eulalio, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+screening+identifies+miRNAs+inducing+cardiac+regeneration.">Functional screening identifies miRNAs inducing cardiac regeneration.</a> Nature. 492 (7429), 376-381 (2012).</li><li>Di, S. V., Giacca, M., Capogrossi, M. C., Crescenzi, M., Martelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+cyclin-dependent+kinase+inhibitors+induces+cardiomyocyte+re-entry+in+the+cell+cycle.">Knockdown of cyclin-dependent kinase inhibitors induces cardiomyocyte re-entry in the cell cycle.</a> J.Biol.Chem. 286 (10), 8644-8654 (2011).</li><li>Hosoda, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonality+of+mouse+and+human+cardiomyogenesis+in+vivo.">Clonality of mouse and human cardiomyogenesis in vivo.</a> Proc.Natl.Acad.Sci.U.S.A. 106 (40), 17169-17174 (2009).</li><li>Soonpaa, M. H., 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=Assessment+of+cardiomyocyte+DNA+synthesis+in+normal+and+injured+adult+mouse+hearts.">Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.</a> Am.J.Physiol. 272 (1 Pt 2), H220-H226 (1997).</li><li>Ang, K. L., et al. <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> Am.J.Physiol Cell Physiol. 298 (6), C1603-C1609 (2010).</li><li>Engel, F. B., Schebesta, M., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anillin+localization+defect+in+cardiomyocyte+binucleation.">Anillin localization defect in cardiomyocyte binucleation.</a> J Mol Cell Cardiol. 41 (4), 601-612 (2006).</li><li>Bergmann, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Cell+Generation+and+Turnover+in+the+Human+Heart.">Dynamics of Cell Generation and Turnover in the Human Heart.</a> Cell. 161 (7), 1566-1575 (2015).</li></ol>

Es gibt eine Kontroverse darüber, ob Kardiomyozyten können den Zellzyklus und teilen sich nach der Verletzung und während der Gewebehomöostase erneut einzugeben. Werte für den Grundumsatz von Kardiomyozyten wurden im Bereich zwischen 1% und 80% 1 7 angegeben. Auch nach einer kardialen Läsion, die Induktion von Zellzyklusaktivität und die Erzeugung neuer Kardiomyozyten wurde in der Grenzzone berichtet, wobei Werte zwischen 0,0083% 8 und 25 - 4...

Die korrekte Identifizierung von Kardiomyozyten Kerne und der Zellzyklusstatus ist von entscheidender Bedeutung für die Bestimmung der Herzmuskel Umsatz und Regeneration. Dies gilt insbesondere für die Nutzung der Kernmarker wie pHH3, Ki-67, oder Thymidin-Analoga, für die Zellzyklusaktivität identifiziert. Da die proliferative Kapazität von erwachsenen Säugetieren Kardiomyozyten ist sehr klein 1, eine falsche Identifizierung eines Kerns für eine positive Proliferationsmarker eines Kardiomyozyten Kern einen entscheidenden Unterschied in dem Ergebnis eines Proliferationsassay machen könnte. Außerdem sind Kardiomyozyten anfällig für Schwankungen in den Zellzyklus, wie Endoreduplikation und acytokinetic Mitose, die in polyploiden und mehrkernige Zellen führen, anstatt in der Zellteilung. Zu diesem Zweck ist die Interpretation der Antikörperfärbung gegen gemeinsame Zellzyklus-Marker in allen Fällen nicht schlüssig. Hier präsentieren wir Methoden für die lineare forwa<html> rd Anerkennung der Maus Kardiomyozyten und ihre Nuklearität in nativen isolierten Zellen und dicke Gewebeschnitte bei postnatalen und adulten Stadien durch die eindeutige Identifizierung ihrer Kerne. Zu diesem Zweck wird eine transgene Mauslinie mit Kardiomyozyten-spezifische Expression eines Fusionsproteins bestehend aus humanem Histon H2B und mCherry unter der Kontrolle des Promotors Myh6 (Myh6-H2B-MCH) 2 verwendet. Kreuz-Zucht Mauslinie mit einer transgenen Proliferation Indikator Mauslinie, in denen die Expression eines eGFP-Anillin Fusionsprotein unter der Kontrolle des allgegenwärtigen Huhn-Aktin-Promotor mit einem CMV-Enhancer (CAG-eGFP-Anillin), ermöglicht die Bestimmung der Zellzyklus-Status. Das Gerüstprotein Anillin wird in Zellzyklus - aktiven Zellen 3 und seine differentielle subzelluläre Lokalisierung während des Zellzyklus ermöglicht Live-tracking Zellzyklusprogression mit einer hohen Auflösung von M-Phase spezifisch exprimiertef &quot;&gt; 4. Daher kann der doppelt transgenen Mäuse verwendet werden , um zwischen proliferierenden Kardiomyozyten und diejenigen zu unterscheiden , die Zellzyklus - Variationen unterzogen werden . Dies erweist sich als besonders nützlich beim Screening auf die Proliferation induzierenden Substanzen in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="999">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">10 cm petri dish</td>
			<td style="width:331px;">Sarstedt</td>
			<td style="width:119px;">821472</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">100 &#181;m cell strainer</td>
			<td style="width:331px;">Becton Dickinson GmbH/Falcon</td>
			<td style="width:119px;">352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">2,3-Butanedione monoxime (BDM)</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">G20x1 &#189; injection cannula, Sterican</td>
			<td style="width:331px;">Braun, Melsungen</td>
			<td>4657519</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">20 gauge needle</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">301300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">24-well plates</td>
			<td style="width:331px;">Becton Dickinson GmbH/Falcon</td>
			<td style="width:119px;">353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">2-Methyl-butane</td>
			<td style="width:331px;">Carl Roth GmbH + Co. KG</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">37% formaldehyde solution</td>
			<td style="width:331px;">AppliChem GmbH&#160;</td>
			<td>A0936,1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">3-way stopcock</td>
			<td style="width:331px;">B. Braun Medical Inc.</td>
			<td style="width:119px;">16494C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">50 ml syringe</td>
			<td style="width:331px;">B. Braun Medical Inc.</td>
			<td style="width:119px;">8728810F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">70% ethanol</td>
			<td style="width:331px;">Otto Fischar GmbH</td>
			<td style="width:119px;">27669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Alexa-Fluor-conjugated secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td style="width:119px;">115-605-205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Alpha-Aktinin EA-53, Mouse IgG</td>
			<td style="width:331px;">Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">A7811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">CaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Cell Culture Microplate, 96 Well, Half Area</td>
			<td style="width:331px;">Greiner bio-one</td>
			<td style="width:119px;">675986</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Collagenase B</td>
			<td style="width:331px;">Roche</td>
			<td style="width:119px;">11088815001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">confocal microscope Eclipse Ti-E</td>
			<td style="width:331px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">cryostat CM 3050S</td>
			<td style="width:331px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">donkey serum</td>
			<td>Jackson Immuno Research, Suffolk, GB</td>
			<td style="width:119px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:383px;">EDTA</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fetal calf serum</td>
			<td>PromoCell, Heidelberg</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution (4%)</td>
			<td>PanReac AppliChem</td>
			<td style="width:119px;">A3697</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Gelatine from porcine skin, Type A</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td style="width:119px;">G2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">glass coverslips</td>
			<td style="width:331px;">VWR</td>
			<td style="width:119px;">631-0146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Glucose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Heidelberger extension tube</td>
			<td>IMPROMEDIFORM GmbH</td>
			<td style="width:119px;">MF 1833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Heparin-Natrium</td>
			<td style="width:331px;">Ratiopharm</td>
			<td style="width:119px;">5394.02.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">HEPES</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">HistoBond microscope slides</td>
			<td style="width:331px;">Marienfeld</td>
			<td style="width:119px;">0810000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342 (1mg/ml)</td>
			<td>Sigma Aldrich, Taufkirchen</td>
			<td style="width:119px;">B2261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Insulin syringe</td>
			<td>Becton Dickinson GmbH</td>
			<td style="width:119px;">300334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#8217;s ModifiedDulbecco&#8217;s Medium (IMDM)</td>
			<td>Gibco/Life Technologies, Darmstadt</td>
			<td>21980-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">KCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Laminin</td>
			<td style="width:331px;">Corning</td>
			<td style="width:119px;">354221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser Scanning Mikroskop Eclipse Ti</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine RNAiMAX</td>
			<td>Invitrogen/Life Technologies, Darmstadt</td>
			<td>13778075</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:383px;">Mouse IgG Cy5 (donkey)</td>
			<td style="width:331px;">Jackson ImmunoResearch</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">MGCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">microcentrifuge tube</td>
			<td style="width:331px;">Sarstedt</td>
			<td style="width:119px;">72690</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Mini shaker</td>
			<td style="width:331px;">VWR</td>
			<td>12620-940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">mirVana miRNA mimic, hsa-miR199a-3p</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4464066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Biopsy Mold</td>
			<td style="width:331px;">Sakura Finetek/ VWR</td>
			<td>4565</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">M-slide 8-well ibiTreat</td>
			<td style="width:331px;">ibidi</td>
			<td style="width:119px;">80826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">NaCl</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">NaOH</td>
			<td style="width:331px;">Merck Millipore</td>
			<td style="width:119px;">567530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">negative control(scrambled RNA)</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>AM4611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neonatal Heart Dissociation Kit</td>
			<td>Miltenyi Biotech, Bergisch Gladbach</td>
			<td style="width:119px;">130-098-373</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR 4.12.01-4.30.02-64bit</td>
			<td>Nikoninstruments, D&#252;sseldorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non essential amino acids, NEAA</td>
			<td>Gibco/Life Technologies, Darmstad</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Opti-MEM, Reduced Serum Medium</td>
			<td style="width:331px;">Gibco</td>
			<td style="width:119px;">51985-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">P21-siRNA</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">P27-siRNA</td>
			<td style="width:331px;">Ambion/Thermo Fischer Scientific</td>
			<td>4390771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Life Technologies, Darmstadt</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:383px;">Polyvinyl alcohol mounting medium with DABCO&#174;, antifading</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">10981</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">RNase A</td>
			<td style="width:331px;">Qiagen</td>
			<td>1007885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNaseZap</td>
			<td>Invitrogen/Life Technologies, Darmstadt</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">sample containers</td>
			<td style="width:331px;">Vitlab</td>
			<td style="width:119px;">80731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Serological pipette</td>
			<td style="width:331px;">Greiner</td>
			<td style="width:119px;">607180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">software NIS Elements</td>
			<td style="width:331px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Sucrose</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:331px;">Sakura Finetek/ VWR</td>
			<td>25608-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">ToPro3 iodide (642/661)</td>
			<td style="width:331px;">Molecular probes/ThermoFisher Scientific</td>
			<td style="width:119px;">T3605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Tris</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton X</td>
			<td style="width:331px;">Fluka</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton X-100</td>
			<td style="width:331px;">Fluka</td>
			<td style="width:119px;">93418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Trypsin</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Wheat germ agglutinine (WGA) Fluorescein labeled</td>
			<td>Vector Laboratories</td>
			<td>VEC-FL-1021-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">&#945;-actinin antibody</td>
			<td style="width:331px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich, Steinheim</td>
			<td>M3148</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of cell cycle variations and determination of nucleation in postnatal cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

Um die Effekte von siRNAs / miRNAs auf der Zellzyklusaktivität der postnatalen Kardiomyozyten in vitro, Kardiomyozyten von Doppel transgenen Myh6-H2B-mCh / CAG-eGFP-Anillin Mäuse wurden isoliert auf postnatalen Tag 3 (P3) und transfizierte zu analysieren mit Zellzyklusaktivität auslösenden miR199 5, siRNA p27 und siRNA Fzr1. Im Vergleich zur Negativkontrolle (Abbildung 1A), die Bilder von miR199- (Abbildung 1B) und p2...

Watch this Scientific Journal Video about Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes at JoVE.com

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Visualisierung von Zellzyklus-Variationen und Bestimmung der Nukleation in postnatale Kardiomyozyten

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

The overall goal of this procedure is to visualize cell cycle activity in postnatal cardiomyocytes and to determine their nuclearity. This method can help answer key questions in the cardiac regeneration field, such as does a cardiomyocyte really divide or does it just increase its ploidy or become multi-nucleated? The main advantages of this technique are that the M phase of the cell cycle can be visualized in high spatiotemporal resolution and that cardiomyocyte nuclei can be identified by fluorescence.Demonstrating the procedure will be Patricia Freitag, a technician from our laboratory. If using non-homozygous breeding pairs, first identify the transgenic hearts via fluorescent microscopy to check for their EGFP analin and H2B-mCherry expression. EGFP analyin nuclear signals can be most easily detected at the edges of the atria by focusing through its tissue layers.To isolate the cardiomyocytes, place the appropriate number of hearts in 1.5 milliliter reaction tubes containing one milliliter of enzyme mix from a neonatal heart dissociation kit and cut the hearts into small pieces with scissors. Then, transfer the solution into 15 milliliter reaction tubes. Place the tube in an almost horizontal position at 37 degrees celsius for 15 minutes, mixing the tissue five to 10 times with a five milliliter pipette at the end of the incubation.At the end of the third digestion, stop the reaction with 7.5 milliliters of medium two and filter the cell suspension through a 70 micron cell strainer. Rinse the cell strainer with three milliliters of medium two, pooling the wash with the collected cells and collect the cells by centrifugation. Re-suspend the pellet in 500 microliters of medium one for counting.Then, seed one times ten to the fourth cells per well in 120 microliters of medium one in a 96-well flat-bottom plate and centrifuge the cells before their overnight culture in a cell culture incubator. The next day, most of the cardiomyocytes should be beating. Before beginning the transfection, wipe the bench and all of the transfection materials with RNAse decontamination solution to remove any RNAse.When all of the materials are ready, add 20 microliters of freshly prepared siRNA mix to each well and return the cells to the incubator for 48 hours. After two days, replace the supernatant in each well with 120 microliters of medium one and return the cells to the incubator for at least another 24 hours. At the end of the incubation, wash the cells one time with PBS, followed by fixation in 4%formaldehyde solution for 20 minutes.To analyze the cells by confocal video microscopy, transfer the plate to the confocal microscope stage and start the imaging software. Open the A1plus settings and check the channel one, two, three and four boxes. In the pull-down menu, set channel one to DAPI, channel two to eGFP, channel three to RFP, and channel four to Cy5.Click the channel voltage and use the slide bars to set the voltage to 80 for each channel. Use the slide bars to set the offset to zero and click on the home button to set the pinhole to the home position. Set the scan size to 1024 by 1024 pixels in the pull-down menu.Click optimize to open the XYZ size setup window and check the perfect voxel box under the suggested step Z.Select the 20x objective and adjust the laser intensities until the picture is neither under-nor overexposed. When all of the parameters have been set, open the acquire menu. Select scan large image then choose current position is at top left corner under area.Then set the number of fields in X and Y to three by three and click scan. To define the number of cardiomyocyte nuclei, click measure, manual measurement, and counts. Select the nuclei with H2B-mCherry signals, thereby marking and counting them.Then, select the alpha-actinin positive cells to determine the number of cardiomyocytes. To count the G1, S, G2 phase cardiomyocytes with nuclear EGFP analin signals, select measure, manual measurement, and counts. Mark the EGFP analin and H2B-mCherry expressing nuclei with clicking and manually count the cardiomyocytes with the mitosis-specific EGFP analin signals such as contractile rings and mid-bodies.Then, click measure, manual measurement, and counts and click the cardiomyocytes with mitosis-specific EGFP analin signals, cytoplasmatic signals, contractile rings, and mid-bodies. Compared to the negative controls, the miRNA and siRNA transfected cardiomyocytes demonstrate an induction of cell cycle activity. Cardiomyocytes that perform endoreduplication exhibit an exclusively nuclear EGFP analin expression, whereas cardiomyocytes undergoing cell division display EGFP analin expression in M phase typical localizations.After enzymatic digestion of the heart tissue at the Langendorff apparatus, the atria and ventricles can be mechanically separated and analyzed independently, allowing the visualization of H2B-mCherry transgenic ventricular cardiomyocytes with a high number of H2B-mCherry positive bi-nucleated cardiomyocytes. By contrast, the majority of atrial cardiomyocytes are mono-nucleated. Enzymatic digestion does not result in a 100%single-cell population revealing a pattern of cross-striation by alpha-actinin staining, further facilitating the discrimination between bi-nucleated cardiomyocytes as a continuous pattern of cross-striation and cell doublets.To analyze the bi-nucleation index of cardiomyocytes in thick slices, it is necessary to manually scroll through the stack as the nuclei do not necessarily lay within one Z-plane, with wheat germ agglutinin staining permitting the detection of the cell borders. 3D reconstructions of fixed slices of adult transgenic mouse hearts allow the detection and automated counting of hooks, stained, and H2B-mCherry positive nuclei to determine the proportion of cardiomyocyte nuclei under physiological conditions within the tissue. Once mastered, the heart dissociation can be completed in two to three hours if it is performed properly.While attempting this procedure, it is important to remember to work continuously to maintain the viability of the cells throughout the experiment. Following this procedure, it is possible to screen microRNAs or other small substances for their proliferation-inducing effects in postnatal cardiomyocytes.

Research

JoVE Journal

Developmental Biology

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Organotypischen Kulturen für Studien der postnatale Neurogenese

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Forschung

Entwicklungsbiologie

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolierung und Kryokonservierung von neonatalen Rattenkardiomyozyten

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Visualisierung von Chondrozyten-Einlagerung und Directional Verbreitung über Zebrabow klonalen Zellanalyse in der embryonalen Meckel-Knorpel

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Die Differenzierung der Atrial Kardiomyozyten aus pluripotenten Stammzellen Mit dem BMP-Antagonisten Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Die Großproduktion von Kardiomyozyten aus humanen pluripotenten Stammzellen hochreproduzierbarer Kleinmolekül-basierte Differenzierung Protokoll

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Imaging Gelöscht Embryonale und postnatale Hearts bei Einzelzell-Auflösung

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Reifung von humanen Stammzellen abgeleiteten Kardiomyozyten in Biowires Verwendung elektrischer Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Elektrophysiologische Analyse von humanen Pluripotenten Stammzellen-abgeleiteten Kardiomyozyten (hPSC-CMs) unter Verwendung von Multi-Elektroden-Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Bioengineering von humanisierten Knochenmark-Mikroumgebungen in der Maus und ihre Visualisierung durch Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell Cycle Analysis in C. Elegans Keimbahn mit Thymidin-Analog-EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...