Name: a simple and effective method to consistently isolate mouse cardiomyocytes
Uploaded: 2022-11-11T12:30:00
Description: Watch this Scientific Journal Video about A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes at JoVE.com

这项工作得到了美国国立卫生研究院拨款R01 HL114940（Biesiadecki），R01 AG060542（Ziolo）和T32 HL134616（Sturgill和Salyer）的支持。

本研究中执行的所有程序均由俄亥俄州立大学机构动物护理和使用委员会根据NIH指南批准。
1. 溶液制备
注意：缓冲液浓度请参见 表2 。
<ol><li>预隔离（最多在肌细胞分离前 2 周）<ol><li>灌注缓冲液基质：在 1 L 超纯 18.2 MΩ·cm H 2 O 中制备 1L 灌注缓冲液碱（NaCl、KCl、NaH2PO 4、HEPES、葡萄糖和 BDM），在 0.22 μm 无菌过...

<ol>
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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=Myocytes+isolated+from+rejecting+transplanted+rat+hearts+exhibit+a+nitric+oxide-mediated+reduction+in+the+calcium+current.">Myocytes isolated from rejecting transplanted rat hearts exhibit a nitric oxide-mediated reduction in the calcium current.</a> Journal of Molecular and Cellular Cardiology. 33 (9), 1691-1699 (2001).</li><li>Traynham, C. J., 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=Diesterified+nitrone+rescues+nitroso-redox+levels+and+increases+myocyte+contraction+via+increased+SR+Ca(2+)+handling.">Diesterified nitrone rescues nitroso-redox levels and increases myocyte contraction via increased SR Ca(2+) handling.</a> PLoS One. 7 (12), 52005 (2012).</li><li>Nixon, B. R., 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=Combined+troponin+I+Ser-150+and+Ser-23/24+phosphorylation+sustains+thin+filament+Ca(2+)+sensitivity+and+accelerates+deactivation+in+an+acidic+environment.">Combined troponin I Ser-150 and Ser-23/24 phosphorylation sustains thin filament Ca(2+) sensitivity and accelerates deactivation in an acidic environment.</a> Journal of Molecular and Cellular Cardiology. 72, 177-185 (2014).</li><li>Swager, S. 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=Claudin-5+levels+are+reduced+from+multiple+cell+types+in+human+failing+hearts+and+are+associated+with+mislocalization+of+ephrin-B1.">Claudin-5 levels are reduced from multiple cell types in human failing hearts and are associated with mislocalization of ephrin-B1.</a> Cardiovascular Pathology. 24 (3), 160-167 (2015).</li><li>Pinckard, K. 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=A+novel+endocrine+role+for+the+BAT-released+lipokine+12,13-diHOME+to+mediate+cardiac+function.">A novel endocrine role for the BAT-released lipokine 12,13-diHOME to mediate cardiac function.</a> Circulation. 143 (2), 145-159 (2021).</li><li>Roof, S. R., Shannon, T. R., Janssen, P. M., Ziolo, 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=Effects+of+increased+systolic+Ca2++and+phospholamban+phosphorylation+during+beta-adrenergic+stimulation+on+Ca2++transient+kinetics+in+cardiac+myocytes.">Effects of increased systolic Ca2+ and phospholamban phosphorylation during beta-adrenergic stimulation on Ca2+ transient kinetics in cardiac myocytes.</a> American Journal of Physiology-Heart and Circulatory Physiology. 301 (4), 1570-1578 (2011).</li><li>Harris, J. E., 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=Exercise-induced+3'-sialyllactose+in+breast+milk+is+a+critical+mediator+to+improve+metabolic+health+and+cardiac+function+in+mouse+offspring.">Exercise-induced 3'-sialyllactose in breast milk is a critical mediator to improve metabolic health and cardiac function in mouse offspring.</a> Nature Metabolism. 2 (8), 678-687 (2020).</li><li>Roof, S. R., 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=CXL-1020,+a+novel+nitroxyl+(HNO)+prodrug,+is+more+effective+than+milrinone+in+models+of+diastolic+dysfunction-a+cardiovascular+therapeutic:+an+efficacy+and+safety+study+in+the+rat.">CXL-1020, a novel nitroxyl (HNO) prodrug, is more effective than milrinone in models of diastolic dysfunction-a cardiovascular therapeutic: an efficacy and safety study in the rat.</a> Frontiers in Physiology. 8, 894 (2017).</li><li>Milani-Nejad, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+and+large+animal+models+in+cardiac+contraction+research:+advantages+and+disadvantages.">Small and large animal models in cardiac contraction research: advantages and disadvantages.</a> Pharmacology and Therapeutics. 141 (3), 235-249 (2014).</li><li>Harary, I., Farley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+of+single+isolated+beating+heart+cells.">In vitro studies of single isolated beating heart cells.</a> Science. 131 (3414), 1674-1675 (1960).</li><li>Kono, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+collagenases+and+other+proteolytic+enzymes+in+the+dispersal+of+animal+tissues.">Roles of collagenases and other proteolytic enzymes in the dispersal of animal tissues.</a> Biochimica Biophysica Acta. 178 (2), 397-400 (1969).</li><li>Baker, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+apparatus+for+mammalian+heart+perfusion.">An improved apparatus for mammalian heart perfusion.</a> The Journal of Physiology. 115 (1), 30-32 (1951).</li><li>Powell, T., Twist, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+technique+for+the+isolation+and+purification+of+adult+cardiac+muscle+cells+having+respiratory+control+and+a+tolerance+to+calcium.">A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium.</a> Biochemical and Biophysical Research Communications. 72 (1), 327-333 (1976).</li><li>Motayagheni, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+Langendorff+technique+for+mouse+heart+cannulation:+Improved+heart+quality+and+decreased+risk+of+ischemia.">Modified Langendorff technique for mouse heart cannulation: Improved heart quality and decreased risk of ischemia.</a> MethodsX. 4, 508-512 (2017).</li><li>Zhang, Z., 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=An+improved+procedure+for+isolating+adult+mouse+cardiomyocytes+for+epicardial+activation+mapping.">An improved procedure for isolating adult mouse cardiomyocytes for epicardial activation mapping.</a> Journal of Cellular and Molecular Medicine. 25 (24), 11257-11263 (2021).</li><li>Jian, Z., 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=In+vivo+cannulation+methods+for+cardiomyocytes+isolation+from+heart+disease+models.">In vivo cannulation methods for cardiomyocytes isolation from heart disease models.</a> PLoS One. 11 (8), 0160605 (2016).</li><li>Ackers-Johnson, 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=A+simplified,+Langendorff-free+method+for+concomitant+isolation+of+viable+cardiac+myocytes+and+nonmyocytes+from+the+adult+mouse+heart.">A simplified, Langendorff-free method for concomitant isolation of viable cardiac myocytes and nonmyocytes from the adult mouse heart.</a> Circulation Research. 119 (8), 909-920 (2016).</li><li>Weldrick, J. J., Abdul-Ghani, M., Megeney, L. A., Burgon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+the+isolation+of+postnatal+murine+cardiac+myocyte+and+fibroblast+cells.">A rapid and efficient method for the isolation of postnatal murine cardiac myocyte and fibroblast cells.</a> Canadian Journal of Physiology and Pharmacology. 96 (5), 535-539 (2018).</li><li>Myachina, T. A., Butova, X. A., Khohlova, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+Langendorff-free+method+for+isolation+of+cadiomyocytes+from+adult+rat+heart.">A modified Langendorff-free method for isolation of cadiomyocytes from adult rat heart.</a> AIP Conference Proceedings. 2174 (1), 020140 (2019).</li><li>Omatsu-Kanbe, M., Yoshioka, K., Fukunaga, R., Sagawa, H., Matsuura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+antegrade+perfusion+method+for+isolating+viable+single+cardiomyocytes+from+neonatal+to+aged+mice.">A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal to aged mice.</a> Physiological Reports. 6 (9), 13688 (2018).</li><li>Omatsu-Kanbe, M., Fukunaga, R., Mi, X., Matsuura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+antegrade+perfusion+method+for+cardiomyocyte+isolation+from+mice.">An antegrade perfusion method for cardiomyocyte isolation from mice.</a> Journal of Visualized Experiments. (171), e61866 (2021).</li><li>Bers, D. M., Patton, C. W., Nuccitelli, 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+practical+guide+to+the+preparation+of+Ca2++buffers.">A practical guide to the preparation of Ca2+ buffers.</a> Methods in Cell Biology. 99, 126 (1994).</li><li>Grosso, D. S., Frangakis, C. J., Carlson, E. C., Bressler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+myocytes+from+the+adult+rat+heart.">Isolation and characterization of myocytes from the adult rat heart.</a> Preparative Biochemistry. 7 (5), 383-401 (1977).</li><li>Thum, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butadione+Monoxime+increases+the+viability+and+yield+of+adult+cardiomyocytes+in+primary+cultures.">Butadione Monoxime increases the viability and yield of adult cardiomyocytes in primary cultures.</a> Cardiovascular Toxicology. 1 (1), 61-72 (2001).</li><li>Wolska, B. M., Solaro, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+adult+mouse+cardiac+myocytes+for+studies+of+contraction+and+microfluorimetry.">Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry.</a> American Journal of Physiology. 271 (3), 1250-1255 (1996).</li></ol>

我们的无朗根多夫心肌细胞分离技术的主要优点是，它不需要插管朗根道夫器械，从而限制缺氧和缺血时间。与经典的朗根道夫技术不同，通常需要几分钟才能去除，清洁和悬挂心脏，通常会导致心肌细胞缺血性损伤，我们的方法包括通过冰冷的清除溶液进行体内血液清除。冰冷的清除缓冲液含有乙二胺四乙酸（EDTA），不可逆地螯合二价阳离子以有效去除钙，使其成为极好的抗凝剂...

几十年来，心脏生物学文献中的一个重要思想是分子作用机制。必须建立作用机制才能发表可靠的研究。确定分子机制的成熟策略是分离的心肌细胞研究，这需要高质量的心肌细胞才能获得可靠的数据。对心肌细胞进行的细胞和分子实验以确定作用机制是研究收缩1、电生理学2、钙 （Ca 2+） 循环3、肌纤维Ca 2+ 敏感性4、细胞骨架5、代谢6、激素的影响7、信号分子8、药物研究9 的金标准等。小鼠因其易于基因操作、体积小、寿命相对较短、成本低等优点，成为大多数心脏生物学实验的首选物种10。然而，高质量小鼠心肌细胞的可靠分离在目前的技术中并非易事。
实验室分离心肌细胞已有近70年的历史11。几乎所有分离心肌细胞的技术都依赖于通过各种酶（胶原酶、蛋白酶、胰蛋白酶等）对心脏的消化。在早期（1950 年代至 1960 年代），采用了块法，其中包括取出心脏，切成更小的碎片并与胶原酶/蛋白酶/胰蛋白酶12 在溶液中孵育。在 1970 年代，实验室实施了改进的“Langendorff”方法13，该方法使用基于冠状动脉灌注的分离技术（通过 Langendorff 装置用酶逆行灌注）分离心肌细胞;该技术仍然是今天该领域肌细胞分离的主要方法，~50年后14，15，16。最近的工作已转向在体内插管心脏，以限制缺氧时间和缺血损伤，从而获得卓越的心肌细胞分离（更好的产量和更高的质量）17。最近，这已经演变成在体内进行无朗根多夫的心脏灌注18，19，20，21，22。我们已经在Ackers-Johnson等人18技术的基础上发展了无Langendorff心肌细胞分离技术，并从以前的许多分离技术中采用了各种组件。这些关键的适应措施包括注射冰冷的清除缓冲液和结合支撑平台以稳定针头，从而减少对心脏的操作。该技术还详细介绍了注射缓冲液的温度控制（37°C），由于EDTA灌注较少，因此减少了体内注射和消化之间的时间，如先前发表的18所示。通过减少心脏的操作，从而最小化穿刺部位的大小，可以获得冠状动脉的彻底和持续灌注。我们还通过二次块法消化、注射的清除缓冲液中的EDTA量以及改变pH值来改进该技术。与使用Langendorff设备相比，我们描述的技术更可靠，更高效，并且不需要大量的培训/实践（表1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cc Bd Luer-Lok Syringe</td>
			<td>Fisher Sci</td>
			<td>14-827-52</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Pyrex Low-Form Beaker</td>
			<td>Cole-Palmer</td>
			<td>UX-34502-01</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL polypropylene cap glass media storage bottle</td>
			<td>DWK Life Sciences</td>
			<td>UX-34523-00</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL Round-Bottom Polypropylene Test Tubes With Cap</td>
			<td>Fisher Sci</td>
			<td>14-959-11B</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione Monoxime</td>
			<td>Sigma</td>
			<td>B0753</td>
			<td>&#62;98%</td>
		</tr>
		<tr>
			<td>3 cc BD Luer-Lok Syringe</td>
			<td>Fisher Sci</td>
			<td>14-823-435</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm glass bottom dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.0-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL BD Syringe without Needle</td>
			<td>Fisher Sci</td>
			<td>13-689-8</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Centrifuge Tubes</td>
			<td>Cole-Palmer</td>
			<td>EW-22999-84</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AIMS Space Gel Heating Pad</td>
			<td>Fisher Sci</td>
			<td>14-370-223</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide 27 G X 1/2&#34; Hypodermic Needles</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A3803</td>
			<td>Heat shock fraction, lyophilized powder, essentially fatty acid free, &#62;98%</td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>Suitable for cell culture, &#62;99.5%</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Fisher Sci</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Sci</td>
			<td>AAA1071336</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 100 mm TC-treated Cell Culture Dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>R&#38;D Systems (Bio-techne)</td>
			<td>S11195</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Isotemp Heated Immersion Circulators</td>
			<td>Fisher Sci</td>
			<td>13-874-432</td>
			<td></td>
		</tr>
		<tr>
			<td>Hartman Mosquito Hemostatic Forceps</td>
			<td>World Precision Instruments</td>
			<td>15921</td>
			<td></td>
		</tr>
		<tr>
			<td>Hausser Scientific Hy-Lite Counting Chamber Set</td>
			<td>Fisher Sci</td>
			<td>02-671-11</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>&#62;99.5%</td>
		</tr>
		<tr>
			<td>Labeling Tape</td>
			<td>Fisher Sci</td>
			<td>15-901-10R</td>
			<td></td>
		</tr>
		<tr>
			<td>Legato 100 Syringe Pump</td>
			<td>kdScientific</td>
			<td>788100</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutathione</td>
			<td>Fisher Sci</td>
			<td>ICN19467980</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TH Research Grade</td>
			<td>Sigma</td>
			<td>5401135001</td>
			<td>High thermolysin concentration</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>Fisher Sci</td>
			<td>MT10060CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Invitrogen</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin</td>
			<td>Corning</td>
			<td>354232</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Fisher Sci</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>Precision Digital Reciprocating Water Bath</td>
			<td>ThermoFisher Scientific</td>
			<td>TSCIR19</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>Suitable for cell culture</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
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a simple and effective method to consistently isolate mouse cardiomyocytes

对产生高质量心肌细胞的可重复但技术上简单的方法的需求对于心脏生物学研究至关重要。心肌细胞的细胞和分子功能实验（例如收缩、电生理学、钙循环等）是建立疾病机制的金标准。小鼠是功能实验的首选物种，所描述的技术专门用于分离小鼠心肌细胞。以前需要朗根道夫器械的方法需要高水平的训练和主动脉插管的精度，通常会导致缺血。该领域正在转向无Langendorff的分离方法，这些方法简单，可重复，并产生用于生理数据采集和培养的活肌细胞。与主动脉插管相比，这些方法大大减少了缺血时间，并导致可靠地获得心肌细胞。我们对无Langendorff方法的适应包括使用冰冷的清除溶液进行初始灌注，使用稳定平台以确保灌注过程中针头稳定，以及额外的消化步骤，以确保可靠地获得用于功能测量和培养的心肌细胞。这种方法简单快捷，几乎不需要技术技能。

在确定隔离是否成功时，有几个要素需要检查。首先，心肌细胞必须是杆状的，没有膜泡，例如 图1中分离的细胞。典型的分离将产生~80%的肌细胞呈杆状。如果分离产生的杆状细胞低于50%，则认为分离不成功，不使用心肌细胞。最后，心肌细胞应该是静止的。自发收缩的肌细胞表现出 Ca2+ 不耐受，并且会产生不可靠的数据。在使用上述技术进行的所有分离中，几乎所有分离?...

Watch this Scientific Journal Video about A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes at JoVE.com

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

心脏病学细胞和分子功能实验的金标准是心肌细胞。本文介绍了对非朗根道夫技术分离小鼠心肌细胞的适应性。

一种持续分离小鼠心肌细胞的简单有效的方法

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

与最常用的朗根道夫仪器技术相比，这种技术更可靠、更高效，不需要大量的练习或培训。该技术的主要优点是它限制了缺血时间，因为初始灌注发生在体内。在执行这种技术时，必须避免歧管中的气泡，并避免在消化过程中心脏过度运动，这些会降低产量。展示这项技术的将是Mark Ziolo博士实验室的研究生Sarah Sturgill。首先使用新鲜制备的灌注缓冲液清除温控歧管。为此，请拧入 27 号针并使用鲁尔锁连接器清除气泡。确保没有气泡残留。为了分离心肌细胞，暴露完全麻醉小鼠的胸骨，并在中线的外侧，通过肋骨和腋窝近端切开。然后切开横膈膜，确保浅切口以避免心脏。用止血器夹住胸骨，并将肋骨向后折叠以露出胸腔。轻轻地从心脏上取出心包，然后切开紧邻心脏远端的下腔静脉。使用 27 号针头在一分钟内将三毫升冰冷的清除缓冲液注入心脏的右心室。接下来，用镊子握住心脏。将其从身体上拉开，并尽可能多地暴露主动脉。然后使用止血器夹住升主动脉并切除心脏。快速将夹紧的心脏转移到含有10毫升温灌注缓冲液的聚丙烯培养皿的盖子上。将夹紧的心脏放入支撑平台，并使用连接到歧管的稳定 27 号针头在 37 摄氏度下将 10 毫升温控灌注缓冲液注入左心室，持续 5 分钟。接下来，将夹紧的心脏和支持平台转移到带有约五毫升消化缓冲液的培养皿中。将输入注射器更换为含有25毫升消化缓冲液的50毫升注射器。注射前清除歧管中的任何气泡和剩余的灌注缓冲液。小心地将针头更换到左心室心尖的相同针头位置。在灌注泵的帮助下，使用50毫升注射器将37摄氏度的消化缓冲液注入左心室15分钟。用校准为喷射37摄氏度溶液的水套控制溶液的温度。从心房取出心室，然后将其转移到 10 毫升烧杯中。在烧杯中加入三毫升消化缓冲液，用锋利的剪刀将心室切成大块。用铝箔盖住烧杯，然后将其放入预热至37摄氏度的摇动水浴中五分钟。弃去上清液后，将组织块重悬于三毫升消化缓冲液中，研磨约四分钟或直到获得均匀的混合物。完成后，使用50微米尼龙细胞过滤器将细胞过滤到70毫升聚丙烯管中。用三毫升消化缓冲液清洗尼龙细胞过滤器。将滤液放回14毫升圆底聚丙烯管中并离心。弃去上清液，然后将沉淀重悬于三毫升终止缓冲液中。向细胞悬液中加入54微升100毫摩尔氯化钙原液，使最终氯化钙浓度为1.8毫摩尔。让活细胞沉淀10分钟，并除去含有死细胞的上清液。将沉淀重悬于储存溶液中后，细胞可用于功能实验和培养。分离细胞的明场显微镜图像显示，小鼠心肌细胞的分离产生了没有膜泡或圆角边缘的杆状静止细胞。这些细胞显示出大约80%的产量和高存活率，决定了分离技术的成功。为了成功分离，最重要的是从歧管中去除气泡并将针头插入心脏的同一孔中。一旦分离，心肌细胞可用于细胞和分子功能实验。

Research

JoVE Journal

Medicine

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

一个型号的不安流致动脉粥样硬化小鼠颈动脉部分结扎术和颈动脉内皮细胞的RNA分离的简单方法

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

科研

医学

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

A Simple Method of Mouse Lung Intubation

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A Modified Precipitation Method to Isolate Urinary Exosomes

一种改进沉淀法来隔离尿外来体

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

A Simple Critical-sized Femoral Defect Model in Mice

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

一个简单的设备，迅速准备小鼠肠的全坐骑

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

一个简单的机械过程来创建在小鼠角膜缘干细胞缺乏症

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

胰腺管输液：药物和病毒输送的有效和选择性方法

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

3D 超声成像: 快速、经济高效的肌骨骼组织形态分析

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...