Name: analyzing oxygen consumption rate in primary cultured mouse neonatal cardiomyocytes using an extracellular flux analyzer
Uploaded: 2019-02-13T13:00:00
Description: Watch this Scientific Journal Video about Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer at JoVE.com

我们要感谢罗斯实验室和墨菲实验室的所有成员。这项工作得到了美国心脏协会 (14sdg17790005) 对 yc. nih (hl115933, hl127806) 和 va 优异 (bx003260) 至 r. s. r. 的支持。

对于使用新生鼠, 请参考当地大学/研究所动物护理计划规定的指南, 并遵守自己的机构和其他适当规定。本协议中描述的所有方法都已得到加州大学圣地亚哥分校动物护理和使用机构委员会 (iacuc) 的批准, 并遵守联邦和州法规。
1. 试剂的制备
<ol><li>准备25 毫升的预处理溶液: hbss (不含 ca2 +和 mg2 +) 补充胰蛋白酶 (0.5 mg/ml)。使用0.22μm 过滤器对溶液?...

<ol>
	<li>Stanley, W. C., Recchia, F. A., Lopaschuk, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+substrate+metabolism+in+the+normal+and+failing+heart.">Myocardial substrate metabolism in the normal and failing heart.</a> Physiological Reviews. 85 (3), 1093-1129 (2005).</li><li>Wang, K., 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=Mitochondria+regulate+cardiac+contraction+through+ATP-dependent+and+independent+mechanisms.">Mitochondria regulate cardiac contraction through ATP-dependent and independent mechanisms.</a> Free Radical Research. , 1-10 (2018).</li><li>Kolwicz, S. C., Purohit, S., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+metabolism+and+its+interactions+with+contraction,+growth,+and+survival+of+cardiomyocytes.">Cardiac metabolism and its interactions with contraction, growth, and survival of cardiomyocytes.</a> Circulation Research. 113 (5), 603-616 (2013).</li><li>Rosca, M. G., Hoppel, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+heart+failure.">Mitochondrial dysfunction in heart failure.</a> Heart Failure Reviews. 18 (5), 607-622 (2013).</li><li>Wai, 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=Imbalanced+OPA1+processing+and+mitochondrial+fragmentation+cause+heart+failure+in+mice.">Imbalanced OPA1 processing and mitochondrial fragmentation cause heart failure in mice.</a> Science. 350 (6265), aad0116 (2015).</li><li>Bayeva, M., Gheorghiade, M., Ardehali, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+as+a+therapeutic+target+in+heart+failure.">Mitochondria as a therapeutic target in heart failure.</a> Journal of American College Cardiololgy. 61 (6), 599-610 (2013).</li><li>Camara, A. K., Bienengraeber, M., Stowe, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+approaches+to+protect+against+cardiac+ischemia+and+reperfusion+injury.">Mitochondrial approaches to protect against cardiac ischemia and reperfusion injury.</a> Frontiers in Physiology. 2, 13 (2011).</li><li>Brown, D. 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=Expert+consensus+document:+Mitochondrial+function+as+a+therapeutic+target+in+heart+failure.">Expert consensus document: Mitochondrial function as a therapeutic target in heart failure.</a> Nature Reviews Cardiology. 14 (4), 238-250 (2017).</li><li>Zhou, P., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recounting+Cardiac+Cellular+Composition.">Recounting Cardiac Cellular Composition.</a> Circulation Research. 118 (3), 368-370 (2016).</li><li>Parameswaran, S., Kumar, S., Verma, R. S., Sharma, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+culture+-+an+update+on+the+in+vitro+cardiovascular+model+and+future+challenges.">Cardiomyocyte culture - an update on the in vitro cardiovascular model and future challenges.</a> Canadian Journal of Physiology and Pharmacology. 91 (12), 985-998 (2013).</li><li>Judd, J., Lovas, J., Huang, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Culture+and+Transduction+of+Adult+Mouse+Cardiomyocytes.">Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes.</a> Journal of Vissualized Experiments. 10 (114), (2016).</li><li>Gibbs, C. L., Loiselle, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+basal+metabolism.">Cardiac basal metabolism.</a> Japanese Journal of Physiology. 51 (4), 399-426 (2001).</li><li>Mdaki, K. S., Larsen, T. D., Weaver, L. J., Baack, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+Related+Bioenergetics+Profiles+in+Isolated+Rat+Cardiomyocytes+Using+Extracellular+Flux+Analyses.">Age Related Bioenergetics Profiles in Isolated Rat Cardiomyocytes Using Extracellular Flux Analyses.</a> PLoS One. 11 (2), e0149002 (2016).</li><li>Neary, M. 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=Hypoxia+signaling+controls+postnatal+changes+in+cardiac+mitochondrial+morphology+and+function.">Hypoxia signaling controls postnatal changes in cardiac mitochondrial morphology and function.</a> Journal of Molecular and Cellular Cardiology. 74, 340-352 (2014).</li><li>Hill, B. G., Dranka, B. P., Zou, L., Chatham, J. C., Darley-Usmar, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+bioenergetic+reserve+capacity+in+response+to+cardiomyocyte+stress+induced+by+4-hydroxynonenal.">Importance of the bioenergetic reserve capacity in response to cardiomyocyte stress induced by 4-hydroxynonenal.</a> Biochemical Journal. 424 (1), 99-107 (2009).</li><li>Sreejit, P., Kumar, S., Verma, R. S. <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+protocol+for+primary+culture+of+cardiomyocyte+from+neonatal+mice.">An improved protocol for primary culture of cardiomyocyte from neonatal mice.</a> In vitro Cellular &amp; Developmental Biology - Animal. 44 (3-4), 45-50 (2008).</li><li>. AVMA Guidelines for the Euthanasia of Animals: 2013 Edition Available from: <a target="_blank" href="https://www.avma.org/KB/Policies/Documents/euthanasia.pdf">https://www.avma.org/KB/Policies/Documents/euthanasia.pdf</a> (2013)</li><li>Manso, A. 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=Talin1+has+unique+expression+versus+talin+2+in+the+heart+and+modifies+the+hypertrophic+response+to+pressure+overload.">Talin1 has unique expression versus talin 2 in the heart and modifies the hypertrophic response to pressure overload.</a> Journal of Biological Chemistry. 288 (6), 4252-4264 (2013).</li><li>Wang, G. W., Kang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+doxorubicin+toxicity+in+cultured+neonatal+mouse+cardiomyocytes+with+elevated+metallothionein+levels.">Inhibition of doxorubicin toxicity in cultured neonatal mouse cardiomyocytes with elevated metallothionein levels.</a> Journal of Pharmacology and Experimental Therapeutics. 288 (3), 938-944 (1999).</li><li>Divakaruni, A. S., 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=Thiazolidinediones+are+acute,+specific+inhibitors+of+the+mitochondrial+pyruvate+carrier.">Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (14), 5422-5427 (2013).</li><li>Rogers, G. W., 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=High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PLoS One. 6 (7), e21746 (2011).</li><li>Sansbury, B. E., Jones, S. P., Riggs, D. W., Darley-Usmar, V. M., Hill, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+function+in+cardiovascular+cells:+the+importance+of+the+reserve+capacity+and+its+biological+regulation.">Bioenergetic function in cardiovascular cells: the importance of the reserve capacity and its biological regulation.</a> Chemico-Biological Interactions. 191 (1-3), 288-295 (2011).</li><li>Sharp, W. W., 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=Dynamin-related+protein+1+(Drp1)-mediated+diastolic+dysfunction+in+myocardial+ischemia-reperfusion+injury:+therapeutic+benefits+of+Drp1+inhibition+to+reduce+mitochondrial+fission.">Dynamin-related protein 1 (Drp1)-mediated diastolic dysfunction in myocardial ischemia-reperfusion injury: therapeutic benefits of Drp1 inhibition to reduce mitochondrial fission.</a> FASEB Journal. 28 (1), 316-326 (2014).</li><li>Tigchelaar, W., 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=Hypertrophy+induced+KIF5B+controls+mitochondrial+localization+and+function+in+neonatal+rat+cardiomyocytes.">Hypertrophy induced KIF5B controls mitochondrial localization and function in neonatal rat cardiomyocytes.</a> Journal of Molecular and Cellular Cardiology. 97, 70-81 (2016).</li><li>Graham, B. H., 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+mouse+model+for+mitochondrial+myopathy+and+cardiomyopathy+resulting+from+a+deficiency+in+the+heart/muscle+isoform+of+the+adenine+nucleotide+translocator.">A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator.</a> Nature Genetics. 16 (3), 226-234 (1997).</li><li>Zhao, Y. Y., 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=Defects+in+caveolin-1+cause+dilated+cardiomyopathy+and+pulmonary+hypertension+in+knockout+mice.">Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (17), 11375-11380 (2002).</li><li>Vander Heiden, M. G., 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=Growth+factors+can+influence+cell+growth+and+survival+through+effects+on+glucose+metabolism.">Growth factors can influence cell growth and survival through effects on glucose metabolism.</a> Molecular and Cellular Biology. 21 (17), 5899-5912 (2001).</li><li>Detillieux, K. A., Sheikh, F., Kardami, E., Cattini, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+fibroblast+growth+factor-2+in+the+adult+myocardium.">Biological activities of fibroblast growth factor-2 in the adult myocardium.</a> Cardiovascular Research. 57 (1), 8-19 (2003).</li><li>Wang, 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=The+acute+extracellular+flux+(XF)+assay+to+assess+compound+effects+on+mitochondrial+function.">The acute extracellular flux (XF) assay to assess compound effects on mitochondrial function.</a> Journal of Biomolecular Screening. 20 (3), 422-429 (2015).</li><li>Dai, D. F., 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=Age-dependent+cardiomyopathy+in+mitochondrial+mutator+mice+is+attenuated+by+overexpression+of+catalase+targeted+to+mitochondria.">Age-dependent cardiomyopathy in mitochondrial mutator mice is attenuated by overexpression of catalase targeted to mitochondria.</a> Aging Cell. 9 (4), 536-544 (2010).</li><li>Ritterhoff, J., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+in+cardiomyopathy:+every+substrate+matters.">Metabolism in cardiomyopathy: every substrate matters.</a> Cardiovascular Research. 113 (4), 411-421 (2017).</li><li>Uosaki, H., Taguchi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Gene+Expression+Analysis+of+Mouse+and+Human+Cardiac+Maturation.">Comparative Gene Expression Analysis of Mouse and Human Cardiac Maturation.</a> Genomics, Proteomics &amp; Bioinformatics. 14 (4), 207-215 (2016).</li></ol>

在这项研究中, 我们建立了一个简单的方案, 用于分离和培养小鼠新生儿心肌细胞。通过使用这些心肌细胞, 我们还优化了条件, 以测量耗氧率使用细胞外流量分析仪系统。该协议允许人们使用小鼠新生儿心肌细胞作为模型系统, 以检查各种因素如何改变心脏主要工作细胞的耗氧量, 类似于在完整的器官中测量的东西。我们的协议不同于以前发布的协议16、18.</s...

为了维持持续的心脏收缩功能, 心肌细胞必须保持细胞能量的恒定供应, 主要是 atp1的形式。在心脏中, 大约95% 的 atp 是由线粒体产生的, 主要是通过氧化磷酸化, 表明线粒体在心脏功能2,3 中发挥着至关重要的生物能量作用。支持这一观点的是, 线粒体功能的失调可导致心肌病和心力衰竭4,5。相反, 恢复线粒体功能已被证明可以改善心脏衰竭的心功能 6,7。因此, 研究线粒体生物能量学的机制, 确定心肌细胞线粒体功能的新调节剂, 不仅可以揭示心脏能量产生的机械洞察, 还可以提供导致心脏能量产生的洞察制定治疗心脏病的新治疗靶点 6,8。
与包含肌细胞和非心肌细胞的混合物的整个心脏相比, 心肌细胞培养非常纯净, 非心肌细胞受到的心脏污染最小, 如成纤维细胞和内皮细胞10。此外, 将心肌细胞从新生幼崽中分离, 可以在短时间内培养大量细胞, 而成人心脏中的细胞为 10,11.最重要的是, 原代培养的成年小鼠心肌细胞存活时间短 (例如 24小时), 并在较长的时间点去分化。新生小鼠心肌细胞可以存活并在培养过程中纵 7 天以上 , 使其成为测试药物化合物和基因操作对心肌细胞线粒体功能的影响的理想选择 10 .当然, 成人和新生儿细胞之间存在显著的生物学差异, 但可用于新生儿细胞培养的时间较长, 使其适合许多不同类型的研究, 包括线粒体功能的研究。
迄今为止, 原代培养的新生小鼠和大鼠心肌细胞已被用作研究心脏生物能量 12,13 的模型。近年来, 研究使用细胞外助焊剂分析仪测量耗氧率 (ocr), 并评估小鼠和大鼠新生儿心肌细胞的氧化能力 14,15。与大鼠相比, 小鼠新生儿心肌细胞的细胞活力较低, 变异性较大 16.另外, 研究基因工程小鼠模型细胞的能力也使得老鼠细胞模型非常重要。鉴于 ocr 研究对细胞数量和播种密度非常敏感, 因此需要开发一种可重现、可靠和简单的协议, 以实现一致的细胞产量和活力。
在这里, 我们报告了一个优化的协议, 已经开发, 使用培养的小鼠新生儿心肌细胞与96良好的格式细胞外流量分析仪 ocr 分析。该方案大大提高了检测的重现性。此外, 该协议不仅为 ocr 分析提供了一种新颖、可重复的方法, 而且还可以适应用于其他实验目的的更大尺寸的培养, 例如研究肌纤维功能和细胞内功能可能需要的培养方法信号通路。
特别是, 该协议描述了一个为期一天的程序, 分离和培养新生儿小鼠心肌细胞在96孔细胞培养板。此外, 它还描述了使用细胞外流量分析仪测量耗氧量的过程。所有使用的溶液都经过无菌或无菌过滤。所有工具均采用75% 乙醇消毒。我们为程序的各个部分提供材料表。用于培养心肌细胞, 所有的程序和步骤都是在标准的细胞培养罩中进行的。该协议是为将新生小鼠心脏与一个垃圾 (约8-10 只小狗) 隔离而开发的。然而, 该协议也可用于隔离心肌细胞从多个垃圾。

<table border="0" cellpadding="0" cellspacing="0" style="width:924px;" width="924">
	<tbody>
		<tr height="28">
			<td height="28" style="height:28px;width:269px;">Antimycin A</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">A8674</td>
			<td style="width:288px;">Inhibits complex III of the mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Cell strainer 100 &#956;m pores</td>
			<td style="width:187px;">FALCON</td>
			<td style="width:180px;">352360</td>
			<td style="width:288px;">To capture undigested tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Collagenase type II</td>
			<td style="width:187px;">Worthington</td>
			<td style="width:180px;">LS004176</td>
			<td style="width:288px;">To make collagenase digestion solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">D-Glucose</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">75351</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM high glucose</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">11965-092</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">DMEM without NaHCO3, Glucose, pyruvate, glumanine, and Hepes</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">D5030-10X1L</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone)</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">C2920</td>
			<td style="width:288px;">Uncouples mitochondrial respiration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fetal bovine serum (FBS)</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">26140-079</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Fibronectin from bovine plasma</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">F1141-5MG</td>
			<td style="width:288px;">To make coating solution for tissue culture plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fine scissors</td>
			<td style="width:187px;">Fine Sciences Tools</td>
			<td style="width:180px;">14060-10</td>
			<td style="width:288px;">For dissection of hearts</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Gelatin from porcine skin</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">G-1890</td>
			<td style="width:288px;">To make coating solution for tissue culture plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">HBSS (Hank&#39;s balnced salt solution,without Ca2+, Mg2+)</td>
			<td style="width:187px;">Cellgro</td>
			<td style="width:180px;">21-022-CV</td>
			<td style="width:288px;">To wash hearts and make pre-digestion and collagnase digestion solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">HEPES (1M)</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">15630080</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Horse serum</td>
			<td style="width:187px;">Life technologies</td>
			<td style="width:180px;">26050-088</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">L-Glutamine</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">G-3126</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">M-199</td>
			<td style="width:187px;">Cellgro</td>
			<td style="width:180px;">10-060-CV</td>
			<td style="width:288px;">To make cell culture medium</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:269px;">Moria spoon</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">NC9190356</td>
			<td style="width:288px;">To wash hearts&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Oligomycin</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">75351-5MG</td>
			<td style="width:288px;">Inhibits mitochondrial ATP synthase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">RIPA buffer</td>
			<td style="width:187px;">Fisher scientific</td>
			<td style="width:180px;">89900</td>
			<td style="width:288px;">To lyse the cells for protein assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Rotenone</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">R8875</td>
			<td style="width:288px;">Inhibits complex I of the mitochondria</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Seahorse XFe96 Extracellular Flux 
			Analyzer</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:180px;"></td>
			<td style="width:288px;">Device used to analyze oxygen consumption rate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Seahorse XFe96 FluxPak</td>
			<td style="width:187px;">Agilent</td>
			<td style="width:180px;">102601-100</td>
			<td style="width:288px;">Package of flux analyzer culture plates, sensor cartridges, and calibrant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Sodium pyruvate</td>
			<td style="width:187px;">SIGMA</td>
			<td style="width:180px;">P2256</td>
			<td style="width:288px;">To make mitochodnrial stress test medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Straight scissors</td>
			<td style="width:187px;">Fine Sciences Tools</td>
			<td style="width:180px;">91401-12</td>
			<td style="width:288px;">For dissection of hearts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe filter 0.2 &#956;m size</td>
			<td style="width:187px;"></td>
			<td style="width:180px;"></td>
			<td style="width:288px;">For sterile filtration of digestion medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Trypsin</td>
			<td style="width:187px;">USB Corporation</td>
			<td style="width:180px;">22715 25GM</td>
			<td style="width:288px;">To make pre-digestion solution</td>
		</tr>
	</tbody>
</table>

analyzing oxygen consumption rate in primary cultured mouse neonatal cardiomyocytes using an extracellular flux analyzer

线粒体和氧化代谢是维持心肌功能的关键。研究表明, 线粒体功能障碍是心力衰竭中心脏功能受损的重要因素。相比之下, 恢复有缺陷的线粒体功能可能对改善心脏衰竭患者的心功能有有益的效果。因此, 研究线粒体功能的调节机制和确定新的调节剂可以提供见解, 可用于制定治疗心脏病的新的治疗靶点。在这里, 心脏肌细胞线粒体呼吸使用独特的细胞培养系统进行分析。首先, 优化了一项协议, 以快速分离和培养高生存能力的新生儿小鼠心肌细胞。然后, 用96孔格式的细胞外流量分析仪来评估这些心肌细胞的耗氧率。对于该方案, 我们优化了播种条件, 并证明新生儿小鼠心肌细胞耗氧率可以很容易地评估在细胞外流量分析仪。最后, 我们注意到, 我们的协议可以应用于更大的培养规模和其他研究, 如细胞内信号转导和收缩功能分析。

通过使用所描述的协议, 从第0天的新生幼崽中分离出心脏。获得了 5 x10 5 细胞/幼崽, 并在96个井板的密度为 10 x 10 3、20x 103 或30 x10 3的心肌细胞 (图 2a)。经过一夜的培养, 发现心肌细胞很好地附着在涂层的塑料表面, 很少有未附着的细胞 (与传播的健康的、附着的细胞相比, 未附着的细胞仍然会出现圆形和闪亮的样子) (<s...

Watch this Scientific Journal Video about Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer at JoVE.com

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

细胞外通量分析仪分析原代培养小鼠新生心肌细胞的耗氧率

该方案的目的是说明如何使用小鼠新生儿心肌细胞作为一个模型系统, 以检查各种因素如何改变心脏的耗氧量。

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

该协议允许我们分离和培养高双BBT，只有小鼠心肌细胞知道。为了评估线粒体功能，可以通过实际的银通量分析仪分析心肌细胞的耗氧率。该技术的优点之一是，心肌细胞的耗氧量可以轻松地以 96-well 格式以粘附状态测量，从而能够测试具有大量复制的多种条件。该容器为心脏病学研究领域提供了重要的见解。除了耗氧，我们还可以研究其他代谢参数，如灰度和呼吸性环氧。实现心肌细胞的高变异性对于这个实验至关重要。这需要有效的心脏消化。由于新生儿心肌细胞非常脆弱，温柔处理心肌细胞也很重要。在细胞培养罩中，将5ml的HBSS分到6井细胞培养板的每个井中，并放在冰上。此外，将10ml的HPSS加入10cm细胞培养皿，然后在50ml无菌锥形管中准备20ml的尝试素消化液。将所有溶液放在冰上。接下来，提取心脏并立即转移到含有HBVS的无菌细胞培养皿。去除任何残留的肺组织和较大的血管。用柔和的搅拌洗心。随后，用细剪刀将心脏切成8块，用钳子将心脏组织转移到HPSS的6井细胞培养板的一个井中。使用 Moria 勺，在充满 HBSS 的 6 井板中，将其从井转移到井中，清洗心脏组织。然后将心脏组织转移到含有20ml Trypsin的锥形管中，并在4摄氏度的温和搅拌下孵育4小时。在这个过程中，在37摄氏度的水浴中预热胶原酶消化溶液。将含有心脏组织和消化不良溶液的锥形管从4摄氏度转移到细胞培养罩。让心脏组织沉入管底，使用10ml血清移液器去除消化不良溶液。接下来，在管中加入10mlHS。用HPSS重新吸收心脏组织2至3次，使用10ml血清移液器洗净尝试素。吸气HSS和添加10毫升预预热胶酶消化溶液到管与心脏组织。对于第一次消化，在37摄氏度的水浴中用心脏组织孵育管，10分钟不搅拌。之后，将管子转移到细胞培养罩。使用 10ml 血清移液器轻轻重新吸收组织 10 次，使组织重新缝合。这将允许心脏分散，细胞从心脏组织释放。让未消化的组织下沉。将富含心肌细胞的消化溶液转移到新的锥形管中，并立即添加同等数量的细胞培养基，以阻止胶原酶消化。然后加入10ml的胶原酶消化液到管中含有剩余的未消化的心脏组织。对于第二次消化，在37摄氏度的水浴中用心脏组织孵育管，10分钟。重复第一次消化的过程，在停止胶原酶消化之前，将心肌细胞中丰富的消化溶液转移到新的锥形管中。接下来，将无菌细胞过滤器放在新的无菌 50ml 锥形管中。用2至3ml细胞培养器预湿细胞过滤器，并通过细胞过滤器传递细胞。随后，用细胞培养培养剂冲洗细胞过滤器。然后，在180倍重力下，将含有心肌细胞的锥形管离心5分钟。5分钟后，吸进上经剂，在10ml细胞培养培养中重新暂停细胞颗粒。将细胞板到10cm细胞培养盘上，孵育一小时。之后，轻轻搅动盘子。用10ml血清移液器在培养皿上反复移液细胞培养培养，洗掉非粘附细胞，重新暂停细胞。然后，将非粘附细胞转移到一个新的10cm细胞培养皿中，再孵育一小时。一小时后，轻轻搅拌盘子，洗掉非粘附细胞。将心肌细胞转移到新的50ml锥形管中。接下来，在96井细胞培养板的每个井中，通过等50微升的涂层溶液来准备一个96井培养板。如果存在气泡，请使用 20 微升移液器将其清除。在 37 摄氏度下孵育板至少一小时，以便干燥基质涂层。然后，使用血细胞计计算细胞。将细胞板板到外细胞基质涂层96井细胞培养板，密度为每井1万至3万个细胞。将细胞放在细胞中。要预制耗氧测定，请水合通量分析仪传感器盒至少 3 小时，在公用板的每个井中加入 200 微升的校准液。将传感器盒放回实用板，在 37 摄氏度内孵育，无需 CO2 或 O2 补充至少 3 小时。在测定前一小时，轻轻取出细胞培养基，用200微升预预警线粒体应激测试培养基洗涤细胞两次。第二次洗涤后，加入175微升预预警线粒体应激测试介质。然后在37摄氏度下培养细胞，无需二氧化碳或补充氧气。在线粒体压力测试介质中准备3ml的每个测试化合物。使用多通道移液器将每个化合物的 25 微升加载到传感器盒的喷油器端口中。细胞的状态和任何预处理可能影响注射未组联器FCCP导致的最大恢复。细胞对非反应器的敏感性也会改变，因此，优化每个特定治疗的FCCP工作浓度至关重要。随后，建立细胞外通量测定协议并启动程序。首先，将传感器卡蒂里奇放入机器进行校准。校准步骤完成后，更换检测板的校准器。如果需要，在检测后，使用多通道移液器小心丢弃所有检测介质。并且储存细胞培养球蛋白在负20摄氏度，以利用蛋白质测定进行未来的细胞正常化。通过使用所述的规程，心脏从第0天新生儿幼崽分离，心肌细胞在96孔板中每孔10、20或3万个细胞的密度下播种。经过一夜培养，心肌细胞被发现很好地附着在涂层的塑料表面，并且很少有未连接的细胞。在这一点上，自发收缩心肌细胞很容易看到。每井3万个细胞的种子密度在种子细胞扩散一天后出现汇合。心肌细胞在免疫上被免疫，并含有针对心肌细胞特异性标记物沙康α行为素的抗体。如这里所示，大多数细胞显示α作用素的正染色，表明心肌细胞分离的高纯度。此处显示了一个典型的线粒体应激测试方案。线粒体压力测试从氧气消耗率的基准线测量开始，随后注射抑制 ATPA 的 Oligo myosin。然后，注射未联合剂FCCP，以测量最大耗氧率。最后，随着两个电子传输复合抑制剂的注射，线粒体呼吸完全停止，OCR降至最低水平。获得一致且可重现的结果。实现高生存率至关重要。因此，重要的是使用新生儿和轻轻处理心肌细胞的心脏组织。通过使用新的或现有的基因操纵的小鼠线，这种方法可以很容易地转化为数据，可能导致了解新的机制的生物能量调节在心脏。线粒体在心脏功能中起着关键的作用。我们期望这种方法有助于识别新的调节剂和调节氧化代谢的通路，并找到治疗心力衰竭的新治疗靶点。

Research

JoVE Journal

Medicine

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

可视化和肝脏微循环血流量和耗氧量分析：急性肝炎模型中的应用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

科研

医学

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

心率在小鼠中通过无创M型超声心动图测量子宫内

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

A Neonatal Mouse Spinal Cord Compression Injury Model

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

后半圆管法在新生小鼠内耳基因传递中的研究

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

Autonomic Function Following Concussion in Youth Athletes: An Exploration of Heart Rate Variability Using 24-hour Recording Methodology

青年运动员脑震荡后的自主功能: 用24小时记录法探讨心率变异性

Participation in organized sports makes a significant contribution to youth development, but places youth at a higher risk for sustaining a concussion. To ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

白念珠菌实时细胞外通量实时分析的生物能量学研究

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

使用皮肤心肌细胞进行心脏功能的体外评估

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...