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.

동물을 포함하는이 프로토콜의 모든 절차는 본 대학의 윤리적 기준에 따라되었고 과학적인 목적으로 사용되는 동물의 보호에 관한 지침 2010 유럽 의회 / 63 / EU의 가이드 라인을 준수. 출생 후 심근 세포의 세포주기 활동의 체외 시각화 1. <ol><li> 출생 후 심근 해리 <ol><li> 사전 실험 준비 <ol><li> 배양 배지 (IMDM, 1 % 페니실린 / 스트렙토 마이신, 1 % 비 필수 아미노산, 0.1 % ...

<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>

심근 세포가 세포주기를 다시 입력 및 손상 후 조직의 항상성 동안 나눌 수 있는지 논란이있다. 심근의 기본 회전율의 값은 1 % (1) 80 % (7) 사이의 범위에 제시되어있다. 40 % - 7 또한 심장 병변 후 세포주기 활성의 유도 및 새로운 심근 세포의 생성은 0.0083 %, 8 및 25 사이의 값과, 경계 존에보고되었다. 이러한 불일치는 부분적 학...

심근 핵 및 세포주기 상태의 정확한 식별은 심근 회전율 및 재생의 판정을위한 매우 중요하다. 이것은 세포주기 활동을 식별하기위한 예컨대 pHH3, 기 - (67) 또는 티미 딘 유사체 핵 마커의 사용에 특히 그러하다. 성인 포유 동물 심근 세포의 증식 능력은 (1), 증식 분석의 결과에 중요한 차이를 만들 수있는 심근 핵의 확산 마커에 대한 긍정적 인 핵의 잘못된 식별 매우 작은 때문이다. 또한, 심근 배수체 및 다핵 세포가 아닌 세포 분열 될 같은 endoreduplication acytokinetic과 같은 유사 분열 세포주기의 변동,되기 쉽다. 이를 위해, 일반적인 세포주기 마커에 대한 항체 염색의 해석은 모든 경우에 결정적인 아니다. 여기, 우리는 직선 forwa을위한 방법을 제시<html> RD 마우스 심근의 인식과 핵의 명확한 식별하여 출생 후의 성인 단계에서 기본 절연 세포와 두께의 조직 절편에서의 nuclearity. 그 목적을 위해, Myh6 프로모터 (Myh6-H2B-MCH)의 제어하에 인간 히스톤 H2B mCherry과의 융합 단백질의 심근 특이 식 트랜스 제닉 마우스는 2 라인을 사용 하였다. eGFP는-anillin 융합 단백질의 발현이 CMV 인핸서와 유비쿼터스 닭 액틴 프로모터의 제어하에있는 형질 전환 증식 지표 마우스 선이 마우스 라인을 교배 (CAG-eGFP는-anillin)는 허용 세포주기 상태를 결정. 스캐 폴딩 단백질 anillin 특히 활성 셀 3 세포주기에서 발현하고, 세포주기 동안 그 차이 세포 내 지역화 M 위상의 고해상도 실시간 추적 세포주기 진행을 허용EF &quot;&gt; 4. 따라서, 이중 형질 전환 쥐 심근 세포 증식 및 세포주기 변동을 겪는 것과 구별하기 위해 사용될 수있다.이 시험 관내 증식을 유도하는 물질의 스크리닝에 유용 증명한다.

<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.

시험 관내에서 출생 후의 심근 두 번 형질 전환 Myh6-H2B-MCH / CAG-eGFP는-anillin 쥐의 심근 세포의 세포주기 활동에 대한 siRNA를 /의 miRNAs의 효과를 분석하기 위해 출생 후 하루 3 (P3)에 격리하고, 형질 세포주기 활성 유도 miR199 5 P27의 siRNA 및 siRNA를 Fzr1있다. 음성 대조군 (도 1a)와 비교 miR199- (도 1b)와 siRNA를 p27- (도 1C)의</stron...

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.

출생 후 심근 세포의 세포주기 변화의 시각화 및 핵의 결정

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

출생 후 신경 발생의 연구 Organotypic 슬라이스 문화

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

연구

발생학

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

분리 및 신생아 쥐 심근 세포의 냉동 보존

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

배아 멕켈의 연골에 Zebrabow 클론 세포 분석을 통해 연골 세포 인터와 방향 확산의 시각화

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

BMP 길항제 Grem2를 사용하여 다 능성 줄기 세포에서 심방 심근의 차별화

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

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

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

영상은 단일 셀 해상도에서 배아 및 출생 후의 마음을 해제

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

전기 자극을 사용하여 Biowires 인간 줄기 세포 유래 심근 세포의 성숙

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

다중 전극 배열 (MEAs)을 이용한 인간 다 능성 줄기 세포 유래 심근 세포 (hPSC-CMs)의 전기 생리 학적 분석

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

마우스의 인간 골수 미세 환경의 생체 공학 및 실시간 영상화에 의한 시각화

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

티 미 딘 아날로그 듀와 선 충 C. 생식에 세포 주기 분석

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