We would like to thank Dr. Alengo Nyamay'antu and Dr. Ilse Timmerman for their advices about lentiviral packaging. This work is supported by an American Heart Association Scientist Development Grant 10SDG4170137.

<ol>
	<li>Gross, W. O., Schopf-Ebner, E., Bucher, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+the+preparation+of+homogeneous+cultures+of+isolated+heart+muscle+cells.">Technique for the preparation of homogeneous cultures of isolated heart muscle cells.</a> Experimental cell research. 53, 1-10 (1968).</li><li>Toraason, M., Luken, M. E., Breitenstein, M., Krueger, J. A., Biagini, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+toxicity+of+allylamine+and+acrolein+in+cultured+myocytes+and+fibroblasts+from+neonatal+rat+heart.">Comparative toxicity of allylamine and acrolein in cultured myocytes and fibroblasts from neonatal rat heart.</a> Toxicology. 56, 107-117 (1989).</li><li>MacGregor, R. R., Klein, R. M., Bansal, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+of+plasminogen+activator+activity+from+neonatal+rat+heart+cells+is+regulated+by+hormones+and+growth+factors.">Secretion of plasminogen activator activity from neonatal rat heart cells is regulated by hormones and growth factors.</a> Annals of the New York Academy of Sciences. 752, 331-342 (1995).</li><li>Louch, W. E., Sheehan, K. A., Wolska, 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=Methods+in+cardiomyocyte+isolation,+culture,+and+gene+transfer.">Methods in cardiomyocyte isolation, culture, and gene transfer.</a> Journal of molecular and cellular cardiology. 51, 288-298 (2011).</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> The American journal of physiology. , 271-1250 (1996).</li><li>Kaestner, 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=Isolation+and+genetic+manipulation+of+adult+cardiac+myocytes+for+confocal+imaging.">Isolation and genetic manipulation of adult cardiac myocytes for confocal imaging.</a> J Vis Exp. 31 (31), (2009).</li><li>Xu, X., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+adult+rat+heart+myocytes.">Primary culture of adult rat heart myocytes.</a> J Vis Exp. 28 (28), (2009).</li><li>Pinz, I., Zhu, M., Mende, U., Ingwall, J. 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+isolation+procedure+for+adult+mouse+cardiomyocytes.">An improved isolation procedure for adult mouse cardiomyocytes.</a> Cell biochemistry and biophysics. 61, 93-101 (2011).</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.+In+vitro+cellular+&amp;+developmental+biology.">An improved protocol for primary culture of cardiomyocyte from neonatal mice. In vitro cellular &amp; developmental biology.</a> Animal. 44, 45-50 (2008).</li><li>Ehler, E., Moore-Morris, T., Lange, S. <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+Culture+of+Neonatal+Mouse+Cardiomyocytes.">Isolation and Culture of Neonatal Mouse Cardiomyocytes.</a> J Vis Exp. 79 (79), (2013).</li><li>Kass-Eisler, 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=Quantitative+determination+of+adenovirus-mediated+gene+delivery+to+rat+cardiac+myocytes+in+vitro+and+in+vivo.">Quantitative determination of adenovirus-mediated gene delivery to rat cardiac myocytes in vitro and in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 90, 11498-11502 (1993).</li><li>Wasala, N. B., Shin, J. H., Duan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+heart+gene+delivery+vectors.">The evolution of heart gene delivery vectors.</a> The journal of gene medicine. 13, 557-565 (2011).</li><li>Metzger, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications. , (2003).</li><li>Naldini, 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=In+vivo+gene+delivery+and+stable+transduction+of+nondividing+cells+by+a+lentiviral+vector.">In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.</a> Science. 272, 263-267 (1996).</li><li>Zhao, 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=Lentiviral+vectors+for+delivery+of+genes+into+neonatal+and+adult+ventricular+cardiac+myocytes+in+vitro+and+in+vivo.">Lentiviral vectors for delivery of genes into neonatal and adult ventricular cardiac myocytes in vitro and in vivo.</a> Basic research in cardiology. 97, 348-358 (2002).</li><li>Bonci, D., 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=Advanced'+generation+lentiviruses+as+efficient+vectors+for+cardiomyocyte+gene+transduction+in+vitro+and+in+vivo.">Advanced' generation lentiviruses as efficient vectors for cardiomyocyte gene transduction in vitro and in vivo.</a> Gene therapy. 10, 630-636 (2003).</li><li>Murakami, 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=The+FGF+system+has+a+key+role+in+regulating+vascular+integrity.">The FGF system has a key role in regulating vascular integrity.</a> The Journal of clinical investigation. 118, 3355-3366 (2008).</li><li>Nakano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinning-disk+confocal+microscopy+--+a+cutting-edge+tool+for+imaging+of+membrane+traffic.">Spinning-disk confocal microscopy -- a cutting-edge tool for imaging of membrane traffic.</a> Cell structure and function. 27, 349-355 (2002).</li><li>Adams, M. C., 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+high-speed+multispectral+spinning-disk+confocal+microscope+system+for+fluorescent+speckle+microscopy+of+living+cells.">A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells.</a> Methods (San Diego, Calif. 29, 29-41 (2003).</li><li>Wilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinning-disk+microscopy+systems.+Cold+Spring+Harbor+protocols).">Spinning-disk microscopy systems. Cold Spring Harbor protocols).</a> 2010, pdb top88, doi:10.1101/pdb.top88. , (2010).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+microscopy.">ImageJ for microscopy.</a> BioTechniques. 43, 25-30 (2007).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature. 9, 671-675 (2012).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature. 9, 676-682 (2012).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. 32 (32), (2009).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. 63 (63), 10-3791 (2012).</li><li>Mendenhall, A., Lesnik, J., Mukherjee, C., Antes, T., Sengupta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+HIV-+or+FIV-based+lentivector+expression+constructs+and+transduction+of+VSV-G+pseudotyped+viral+particles.">Packaging HIV- or FIV-based lentivector expression constructs and transduction of VSV-G pseudotyped viral particles.</a> J Vis Exp. 62 (62), 10-3791 (2012).</li><li>Durocher, Y., Perret, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+and+high-throughput+recombinant+protein+production+by+transient+transfection+of+suspension-growing+human+293-EBNA1+cells.">High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells.</a> Nucleic acids research. 30, (2002).</li><li>Erbacher, P., Roche, A. C., Monsigny, M., Midoux, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putative+role+of+chloroquine+in+gene+transfer+into+a+human+hepatoma+cell+line+by+DNA/lactosylated+polylysine+complexes.">Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA/lactosylated polylysine complexes.</a> Experimental cell research. 225, 186-194 (1996).</li><li>McSharry, J., Benzinger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+purification+of+vesicular+stomatitis+virus+by+polyethylene+glycol+'precipitation'.">Concentration and purification of vesicular stomatitis virus by polyethylene glycol 'precipitation'.</a> Virology. 40, 745-746 (1970).</li><li>Wulff, N. H., Tzatzaris, M., Young, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monte+Carlo+simulation+of+the+Spearman-Kaerber+TCID50.">Monte Carlo simulation of the Spearman-Kaerber TCID50.</a> Journal of clinical. 2, 10-1186 (2012).</li><li>Kaerber, 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> Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. In: Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. 162. , (1931).</li><li>Sakurai, T., Tsuchida, M., Lampe, P. D., Murakami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+FGF+signaling+is+required+for+Cx43+phosphorylation+and+cardiac+gap+junction+maintenance.">Cardiomyocyte FGF signaling is required for Cx43 phosphorylation and cardiac gap junction maintenance.</a> Experimental cell research. 319, 2152-2165 (2013).</li><li>Ueno, H., Gunn, M., Dell, K., Tseng, A., Williams, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+truncated+form+of+fibroblast+growth+factor+receptor+1+inhibits+signal+transduction+by+multiple+types+of+fibroblast+growth+factor+receptor.">A truncated form of fibroblast growth factor receptor 1 inhibits signal transduction by multiple types of fibroblast growth factor receptor.</a> The Journal of biological chemistry. , 267-1470 (1992).</li><li>Li, Y., Basilico, C., Mansukhani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+transformation+by+fibroblast+growth+factors+can+be+suppressed+by+truncated+fibroblast+growth+factor+receptors.">Cell transformation by fibroblast growth factors can be suppressed by truncated fibroblast growth factor receptors.</a> Molecular and cellular biology. 14, 7660-7669 (1994).</li><li>Murakami, 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=FGF-dependent+regulation+of+VEGF+receptor+2+expression+in+mice.">FGF-dependent regulation of VEGF receptor 2 expression in mice.</a> The Journal of clinical investigation. 121, 2668-2678 (2011).</li><li>Bazan, C., 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=Contractility+assessment+in+enzymatically+isolated+cardiomyocytes.">Contractility assessment in enzymatically isolated cardiomyocytes.</a> Biophysical reviews. , 231-2310 (2012).</li><li>Clark, W. A., Rudnick, S. J., Simpson, D. G., LaPres, J. J., Decker, 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=Cultured+adult+cardiac+myocytes+maintain+protein+synthetic+capacity+of+intact+adult+hearts.">Cultured adult cardiac myocytes maintain protein synthetic capacity of intact adult hearts.</a> The American journal of physiology. , 264-573 (1993).</li></ol>

The authors have nothing to disclose.

从新生大鼠原代心肌早就被用来研究心肌细胞功能的体外 。这个协议描述了用于从幼鼠采用两步酶消化法新生心肌细胞的分离方法，首先用胰蛋白酶O / N消化，在4℃，然后纯化的胶原酶。采用纯化的胶原酶步骤的一个优点在于，同一等级的酶被用于所有隔离。因此，分离的细胞的质量和数量是从实验的实验一致。 给定与腺病毒相关的，如果细胞培养转导的高效率是必需的一个给定的实验中，更高的转导效率，最好是使用一种腺病毒。但是，如果高浓度的转导是没有必要的，慢病毒载体都可以使用。有使用两种腺病毒或慢病毒载体的选择可以更容易地实现适当的transd减税和表达水平不同类型的基因。如果由活细胞成像用共聚焦旋转盘显微镜或IC卡中的荧光强度检测是非常低的慢病毒转导后，使用使用不同的启动子来表达目的基因的慢病毒的另一个转移载体可能会解决此问题。用腺病毒载体，如果腺病毒转导后，世行检测的频带较弱，这可能表明一个低滴度病毒原料。在这种情况下，最好是在细胞繁殖，然后再继续准备较高的滴度腺病毒的解决方案。 这种技术的一个限制是孤立的心肌细胞存活的收益率是不是从实验高度一致地去尝试，用纯化的胶原酶时也是如此。为了进行可重复的成像实验最好是分离的心肌细胞在上清液中的步骤1.2.13选择后计数存活细胞。在协议部分。然后，SE编玻璃底菜的心肌细胞在不同的稀释液。一旦细胞附着，与适当的细胞浓度为实验选择的菜肴计划。 有使用新生儿心肌细胞的许多优点，但也存在一些缺点。主要的缺点是他们从成人的心肌细胞明显的区别。完全分化成心肌细胞是杆状和孤立成人原发性心肌细胞维持其杆状形态，而孤立的新生儿原发性心肌细胞分布在各个方向36。孤立的成人和新生儿心肌细胞之间的表型差异也反映在他们的基因表达37的图案。因此，对于在其中分化的心肌细胞是必需的实验中，一个协议，用于成人心肌细胞的分离，将需要4。 采用重组的腺病毒和慢病毒转导蛋白质进入与纺纱共聚焦显微镜盘组合原代大鼠乳鼠心肌让我们来分析在现场培养原代细胞蛋白质的功能。相对于现有的方法，其利用染引入目的基因导入细胞，这种协议的一个显著优点是，腺病毒和慢病毒是非常有效地吸收由产生的感兴趣的基因在几乎所有的细胞中表达的细胞文化。此外，使用两种不同类型的病毒的基因表达可以更容易地实现适当的转导率和表达水平的多个基因。 siRNA和化学激动剂和拮抗剂也可用于在该系统中，以进一步改动和分析基因的功能和它们编码的蛋白质。 心中的制备是在协议中一个极其重要的一步。尽可能快地清除体内的心。一定要把它们放在冰立刻以保持细胞活力高。另一个重要步骤是高质量的，而且滴度慢病毒/腺病毒的股票的准备。如果质量差的病毒种群的使用，目的基因的转导和表达水平将较低。因此，他们可能不适合成像研究。 这种技术的一个重要应用前景将是在小鼠心肌细胞的使用。试点研究测试是否可用于小鼠乳鼠心肌细胞的分离这种方法已经取得了可喜的成果。由于小鼠心脏相比，这些鼠的相对小的尺寸，更低的组织最初获得的。因此，较少的细胞都使用相同的动物数量，而只是使用更多的老鼠的隔离应消除这一障碍隔绝在一个单一的步骤。隔离小鼠心肌细胞的能力，使研究人员能够研究细胞的基因改造米的隔离功能冰（转基因，基因敲除，敲入）已经产生，以研究目的基因的一种特殊形式。 一个在心血管研究的主要障碍已与调查给定基因的实时表示的心脏组织中在体内的表达和功能相关联的难度。但是隔离功能跳动的心肌细胞，利用病毒转导调节细胞的功能，然后利用共聚焦旋转盘显微镜研究它们在实时的能力，有助于揭示出心肌细胞在体内的作用，弥合这一差距。这种方法提供了对于理解心肌功能在细胞水平上的简化系统。它允许在基因表达的扰动如何改变心肌细胞功能的实时分析。心肌细胞的活细胞成像将有助于进一步深入了解心肌功能。这种见解将导致基本cardiovascula进展Ř研究，导致新的治疗心血管疾病的治疗。

原代大鼠新生心肌早已被用于体外 1调查心肌功能。他们很容易从幼鼠由几个公认的方法2-4隔离开来。最常见的方法使用胶原酶或胰蛋白酶消化之前，细胞分离心脏的结缔组织。研究人员还开发了从成年啮齿动物5-8以及新生小鼠9,10分离心肌细胞的方法。 这个协议描述了从新生大鼠幼仔分离心肌细胞，采用两步酶消化过程的方法。胰蛋白酶是第一次使用O / N在4℃，然后纯化胶原酶在37°C心脏组织用胰蛋白酶O / N的潜伏期在4℃可减少相比，使用顺序孵化在一个温暖的酶液2种方法需要收获细胞的步骤。此外，通过使用纯化的共llagenase而不是粗酶液，批与批之间的变化可以被消除，从而提供增强的可重复性。 特定蛋白质的功能性研究经常采用利用腺病毒11-13和/或14-16的慢病毒的蛋白质表达系统。 [忠告]病毒的生产和操作应根据美国国立卫生研究院的指引进行。 腺病毒不整合到宿主基因组中。它具有转导在大多数细胞类型，包括分裂细胞和非分裂细胞，以及原代细胞和建立的细胞系中非常高的效率。这使得腺病毒的可靠载体用于基因表达。高水平的腺病毒载体编码的蛋白的48小时内下列传导开发，并且它们可以持续数周17。然而，一个缺点是使用的腺病毒蛋白质的表达是一种重组腺的发展novirus是既复杂又费时。这个缺点在部分解释了为什么许多研究人员已经转向慢病毒重组基因的表达。与腺病毒的构建，产生慢病毒结构非常简单快捷。虽然慢病毒通常具有转导的低效率比腺病毒，在两个分裂和非分裂细胞，它们整合到宿主基因组中。因此，转基因的表达对于慢病毒比为腺病毒更稳定。 由于在显微镜领域的技术进步，这是很容易捕捉到细胞中表达重组蛋白的活细胞的图像。这甚至在收购视频速率的速度也是如此。这使得研究者确定如何具体改变在感兴趣的蛋白质在功能上影响实时细胞。共聚焦旋转盘显微镜有，使它成为LIV最佳技术的几个关键特性Ë细胞成像18,19。横河电机旋转的光盘允许更快速图像获取，而在同一时间利用少得多的激光功率比点扫描共聚焦显微镜。这两个特殊的特性是由于旋转的盘，其中包含通过该激光同时传递到许多样品的共焦孔。在采集过程中，磁盘本身旋转快速，持续18-20。通过使用显微镜的自动对焦系统，稳定的焦点被维持在很长一段时间。这使研究人员能够采取有重点的活细胞图像。采集的图像回放的电影文件。图像是使用图像分析软件，如ImageJ的21,22，斐济23，或其它市售软件进行分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1 scissors for decapitation</td>
			<td>WPI</td>
			<td>501749</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>1 fine scissors for heart isolation and chopping</td>
			<td>WPI</td>
			<td>14393</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>2 fine forceps (Dumont No. 5)</td>
			<td>Sigma</td>
			<td>F6521</td>
			<td>Autoclave before use</td>
		</tr>
		<tr>
			<td>Three sterilized 10 cm plastic dishes</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for hearts isolation</td>
		</tr>
		<tr>
			<td>3.5 cm glass bottom culture dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C</td>
			<td>for final plating of cardiomyocytes for future live cell imaging. micro-Dishes from ibidi are an acceptable alternative.</td>
		</tr>
		<tr>
			<td>3.5 cm glass bottom culture dishes</td>
			<td>MatTek</td>
			<td>P35G-0.17-14-C</td>
			<td>for TIRF or high resolution image</td>
		</tr>
		<tr>
			<td>Ethanol solution, 70 % (v/v) in water</td>
			<td>Sigma</td>
			<td>E7148</td>
			<td></td>
		</tr>
		<tr>
			<td>2% gelatin</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>One sterilized 10 cm plastic dish</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for trypsinization</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td>any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>parafilm</td>
			<td>Sigma</td>
			<td>P7543</td>
			<td></td>
		</tr>
		<tr>
			<td>Two 10 cm plastic cell culture dish</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td>for selection</td>
		</tr>
		<tr>
			<td>Auto pipette</td>
			<td>Drummond Scientific&#160;</td>
			<td>4-000-300</td>
			<td>for trituration</td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; Cellometer, automated cell counter</td>
			<td>nexcelom</td>
			<td></td>
			<td>to check and count cells</td>
		</tr>
		<tr>
			<td>&#160; Microscope and Hematocytometer</td>
			<td>any brand</td>
			<td></td>
			<td>to check and count cells</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>invitrogen</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Sanyo</td>
			<td>MCO-19AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>incubating orbital shaker</td>
			<td>Sigma</td>
			<td>Z673129</td>
			<td>to shake heart tissue with collagenase at 37 C at 170-200 rpm</td>
		</tr>
		<tr>
			<td>10 mg/ml BrdU solution</td>
			<td>BD Pharmingen</td>
			<td>550891</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>invitrogen</td>
			<td>41965039</td>
			<td>Mix medium as indicated in the protocol and warm before use</td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>invitrogen</td>
			<td>31095029</td>
			<td>Mix medium as indicated in the protocol and warm before use</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)&#160;</td>
			<td>invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 2 lentiviral transduction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>three packaging plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pRSV-Rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>The lentiviral transfer vector, pLVX-IRES-Puro</td>
			<td>Clontech</td>
			<td>632183</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (serum-free medium)</td>
			<td>invitrogen</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>transfection reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; polyethyleneimine&#8220;Max&#8221;, (Mw 40,000) - High Potency Linear PEI (Equivalent to Mw 25,000 in Free Base Form)&#160;</td>
			<td>Polysciences</td>
			<td>24765-2</td>
			<td>It can be substituted with X-tremeGENE 9 from Roche</td>
		</tr>
		<tr>
			<td>&#160; X-tremeGENE 9</td>
			<td>Roche</td>
			<td>6365779001</td>
			<td>substitute for PEI as transfection reagent</td>
		</tr>
		<tr>
			<td>chloroquine</td>
			<td>Sigma</td>
			<td>C6628</td>
			<td>dissolve in water and make 100 mM stock solution. Inhibition of endosomal acidification can be achieved with 10-100 &#956;M Chloroquine.</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Luer-Lok syringe, sterilized</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 um filters</td>
			<td>Sigma</td>
			<td>F8677</td>
			<td>use only cellulose acetate or polyethersulfone (PES) (low protein binding) filters. Do not use nitrocellulose filters. Nitrocellulose binds surface proteins on the lentiviral envelope and destroys the virus.</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>dissolve in water and make 8mg/ml stock solution, then filter it to sterilize.</td>
		</tr>
		<tr>
			<td>polyethylene glicol 6000</td>
			<td>Sigma</td>
			<td>81260</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 3 Adenoviral transduction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>Some 10 cm cell culture dishes</td>
			<td>Sigma</td>
			<td>CLS430165</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Microplate with lid, flat-bottom, tissue culture, sterile</td>
			<td>BD Falcon</td>
			<td>353072</td>
			<td>for titration</td>
		</tr>
		<tr>
			<td>Multichannel piptte, 10-100 ul, 8-channel</td>
			<td>eppendorf</td>
			<td>3122 000.035</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Section 4 live cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk confocal microspopy</td>
			<td>PerkinElmer</td>
			<td>L7267000</td>
			<td></td>
		</tr>
		<tr>
			<td>a temperature controlled chamber</td>
			<td>any brand</td>
			<td></td>
			<td>to keep temperature at 37 C</td>
		</tr>
		<tr>
			<td>a CO2 environmental system</td>
			<td>any brand</td>
			<td></td>
			<td>optional to maintain CO2 concentration optimal</td>
		</tr>
		<tr>
			<td>Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160; CO2 Independent Medium, No Glutamine</td>
			<td>invitrogen</td>
			<td>18045-054&#160;&#160;</td>
			<td>for long time time-lapse imaging</td>
		</tr>
		<tr>
			<td>&#160; DMEM, High Glucose, HEPES, no Phenol Red&#160;</td>
			<td>invitrogen</td>
			<td>21063-029</td>
			<td>for long time time-lapse imaging</td>
		</tr>
	</tbody>
</table>

live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy

原代大鼠新生心肌细胞是有用的基本体外心血管研究，因为它们可以很容易地分离出大量的单一步骤。由于在显微镜技术的进步是相对容易捕捉到活细胞的图像进行调查的实时有关的光毒性的细胞的细胞事件以最小的关注的目的。本协议描述了如何利用共聚焦旋转盘显微镜下慢病毒和腺病毒转导调节细胞的特性采取原代大鼠乳鼠心肌细胞的活细胞慢速拍摄的图像。两种不同类型的病毒的应用可以更容易地实现合适的转导率和表达水平的两个不同的基因。以及聚焦活细胞图像可以使用显微镜的自动对焦系统，可以保持稳定的聚焦长的时间段来获得。应用这种方法，外源性工程师的功能表达在培养的原代细胞编蛋白进行分析。此外，该系统可用于通过化学式调制器的使用的siRNA以及检查基因的功能。

为了说明的技术中，慢病毒编码EGFP（增强型绿色荧光蛋白）标记的Cx43（连接蛋白43）或突变的Cx43蛋白32，来表达EGFP-标记的蛋白质在细胞和腺病毒编码FGFR1DN（成纤维细胞生长因子受体1显性失活）被用来关闭FGF信号在细胞33-35。三天之后的心肌细胞的分离，该分离的心肌细胞转导的慢病毒，以表达EGFP-标记的蛋白质在细胞内。然后后的慢病毒转导后3天，分离的心肌细胞进一步转导的腺病毒编码FGFR1DN消除FGF信号中的单元格。允许足够的时间让细胞以表达转导的外源基因后，共聚焦旋转盘显微镜被用来捕捉活细胞的图像。采集的图像回放的电影文件，并使用图像分析软件进行分析。如需更详细的代表性略去结果请参见Sakurai 等 32。 以下的慢病毒转导是通过共聚焦旋转盘镜72小时以下的慢病毒转导的图1A-B证实了EGFP的表达标记蛋白。 FGFR1DN由腺病毒转导的表达由世界银行和国际商会腺病毒转导图1C-D的24小时后得到了证实。腺病毒除了3.5厘米菜肴的时间被定义为在时间0和时间推移使用一个腔室的加热器，以保持温度在37˚C.被获取的图像具有40倍的空气NA 0.9的物镜，每5分钟6小时在采取了通过共聚焦旋转盘显微镜初级心肌细胞的EGFP-标记蛋白的时间推移图像示于补充影1-2。 在情况下，温度平衡是不够的，还是多采集模式是不正常，拍摄的图像时，工作正常的自动对焦系统稳定保持焦点可能会移动在xy平面补充动画1。然而，这并没有发生。如图补充电影2，当温度平衡是适当的和被收购的兴趣只有一个区域，拍摄的图像质量非常高的。 心肌细胞的搏动被后以证实细胞活力长期的时间推移活细胞成像评估。即使经过16小时的时间推移成像EGFP表达的心肌细胞保持其功能性和节拍有节奏地补充电影3。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51666/51666fig1highres.jpg" width="500" /> 下面的慢病毒或腺病毒转导图1。确认蛋白的表达。 </strong&gt;的（A）隔离乳鼠心肌细胞表达Cx43蛋白-WT-EGFP转导后3天转用Ad-FGFR1DN。另见补充电影1（B）隔离乳鼠心肌细胞表达Cx43-S325/328/330E（3SE）-EGFP转导后3天转用Ad-FGFR1DN。继转导后，国际刑事法院24小时检测腺病毒转导又见补充电影2（C）的蛋白表达。 FGFR1DN蛋白的以下通过转导后WB 24小时检测到腺病毒转导的血凝素（HA）蛋白标记的FGFR1DN用常规IC卡的方法用抗-HA抗体检测，α-辅肌动蛋白被用来作为标记的心肌细胞（D）的表达。 HA-标记的FGFR1DN用抗HA抗体检测由WB，β-微管蛋白用作上样对照。 大鼠离体新生车补充电影1。时间推移成像<html> diomyocytes表达Cx43蛋白-WT-EGFP转用Ad-FGFR1DN的时间推移图像被收购与40倍的物镜，每5分钟6小时用共焦纺丝磁盘显微镜。采集的图像进行回放作为一部电影每秒10帧。这部影片已被修改Sakurai 等 32。 <a href="https://www.jove.com/files/ftp_upload/51666/s_movie1_2.mov" target="_blank">请点击此处观看该视频。</a> 补充电影2。离体大鼠乳鼠心肌细胞表达Cx43-S325/328/330E-EGFP转用Ad-FGFR1DN时间推移成像。被收购的时间推移图像用40倍物镜6小时，每隔5分钟用共聚焦旋转盘显微镜。采集的图像进行回放作为一部电影每秒10帧。这部影片已被修改Sakurai 等 32。"目标="_blank"&gt;请点击此处观看该视频。 离体大鼠乳鼠心肌细胞补充电影3。时间推移成像回放时的实际速度。离体大鼠乳鼠心肌细胞表达Cx43蛋白-EGFP转导与被收购AD-FGFR1DN用40倍物镜每200毫秒10的时间推移图像秒，共聚焦旋转盘显微镜下16小时的时间推移收购。采集的图像进行回放的电影在每秒5张的实际速度。 <a href="https://www.jove.com/files/ftp_upload/51666/s_movie3.MOV" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy at JoVE.com

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

live-cell-imaging-primary-rat-neonatal-cardiomyocytes-following

Universidad San Sebastian

Research

JoVE Journal

Biology

13.5K 次观看.  Max-Planck-Institute for Molecular Biomedicine and Institute of Cell Biology. This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

视频: Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

分离培养和成年小鼠心肌细胞转导

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

活细胞成像和血管紧张素的三维分析受体1a型贩运转染人胚肾细胞使用共聚焦显微镜

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, misunderstood, or misinterpreted by the fixed-cell analysis. While the fixed cell imaging and analysis is robust and sufficient to observe cellular steady-state, it can be limited in defining a temporal order of events at the cellular level and is ill-equipped to assess the transient nature of dynamic processes including mitotic progression. In contrast, live cell imaging is an eloquent tool that can be used to observe cellular processes at the single-cell level over time and has the capacity to capture the dynamics of processes that would otherwise be poorly represented in fixed cell imaging. Here we describe an approach to generate cells carrying fluorescently labeled markers of chromatin and microtubules and their use in live cell imaging approaches to monitor metaphase chromosome alignment and mitotic exit. We describe imaging-based techniques to assess the dynamics of spindle formation and mitotic progression, including the identification of cells at various stages in mitosis, identification&#160;and tracking of mitotic defects, and analysis of spindle dynamics and mitotic cell fate following the treatment with mitotic inhibitors.

Here we present a protocol to assess the dynamics of spindle formation and mitotic progression. Our application of time-lapse imaging enables the user to identify cells at various stages of mitosis, track and identify mitotic defects, and analyze spindle dynamics and mitotic cell fate upon exposure to anti-mitotic drugs.

活细胞成像，以评估在斜接主轴扰动后元相定时和细胞命运的动态

Live cell time-lapse imaging is an important tool in cell biology that provides insight into cellular processes that might otherwise be overlooked, ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...