Name: isolation of cardiomyocyte nuclei from post-mortem tissue
Uploaded: 2012-07-10T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

我们要感谢马塞洛·托罗用流式细胞仪的协助下。这项研究是由瑞典的心脏和肺脏基金会，欧盟委员会第七框架计划“CardioCell”，瑞典研究理事会，亚洲电影大奖保险和ALF的支持。转播是由德意志研究联合会的支持。

1。分离心脏核<ol><li>外套超离心管（贝克曼离心管＃363664）10毫升1％BSA / PBS涂层解决方案。例管，并让他们旋转管旋转30分钟。删除的涂层解决方案，让空气干燥离心管（每管小鼠心脏单小鼠心脏，交替上升至5小鼠心脏或1克的心脏组织的分析，从不同的物种（如人类）可以处理在一管）。 </li><li>以下所有步骤应在冰上进行。解剖用手术刀从新鲜或冷冻单元的小鼠心脏的左心室。请注意，此协?...

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心肌细胞核的准确鉴定为2,3在心肌再生过程的分析是至关重要的。主要是基于传统技术从新鲜组织分离的心肌细胞细胞外基质蛋白消化酶和随后的间质细胞，低速离心净化。从胚胎干细胞（ESC）的生活心肌的进一步净化可进行免疫标记表面标志物，如9或10 SIRPA线粒体染料，固定的心肌细胞可以识别如肌球蛋白重链（MHC）或核蛋白质，如细 ​​胞质标记GATA4或Nkx2.5。然而，N...

<table border="1"><tbody><tr><td> 1。裂解液 </td></tr><tr><td> 试剂名称 </td></tr><tr><td> 0.32米蔗糖</td></tr><tr><td> 10毫米的Tris-HCl（pH值= 8） </td></tr><tr><td> 5毫米氯化钙2 </td></tr><tr><td> 5 mM的醋酸镁</td></tr><tr><td> 2.0毫米EDTA </td></tr><tr><td> 0.5毫米EGTA </td></tr><tr><td> 1 mM的数码地面电视</td></tr></tbody></table><table border="1"><tbody><tr><td> 2。蔗糖缓冲 </td></tr><tr><td> 试剂名称 </td></tr><tr><td> 2.1 M蔗糖</td></tr><tr><td> 10毫米的Tris-HCl（pH值= 8） </td></tr><tr><td> 5 mM的醋酸镁</td></tr><tr><td> 1 mM的数码地面电视</td></tr></tbody></table><table border="1"><tbody><tr><td> 3。原子核存储缓冲区（国安局加上） </td></tr><tR&gt; <td> 试剂名称 </td></tr><tr><td> 0.44中号蔗糖</td></tr><tr><td> 10毫米的Tris-HCl（pH值= 7.2） </td></tr><tr><td> 70毫米氯化钾</td></tr><tr><td> MgCl 2的 10毫米</td></tr><tr><td> 1.5毫米精</td></tr></tbody></table><table border="1"><tbody><tr><td> 试剂和设备 </td><td> 公司 </td></tr><tr><td>同型兔IgG-CHIP级，＃ab37415 </td><td> abcam </td></tr><tr><td>兔抗-PCM-1抗体，＃HPA023374 </td><td>阿特拉斯抗体</td></tr><tr><td>驴秒。抗体，抗兔Alexa的488福陆公司，＃21206或等效秒。荧光抗体</td><td>生命科技</td></tr><tr><td> DRAQ5 </td><td> biostatus </td></tr><tr><td>细胞过滤30微米，70微米和100微米</td><td> BD公司</td></tr><tr><td>玻璃douncer（40毫升）杵“L” </td><td>厂商VWR（惠顿实业公司） </td></tr><tr><td>的T-25的超Turrax均质</td><td>的IKA德国</td></tr><tr><td>分散工具S25的N-18Ğ </td><td>的IKA德国</td></tr><tr><td>贝克曼阿凡提离心机</td><td>贝克曼</td></tr><tr><td>猎鹰管15毫升和50毫升</td><td>厂商VWR </td></tr><tr><td>贝克曼离心管＃363664 </td><td>贝克曼</td></tr><tr><td> JS13.1自由摆动转子</td><td>贝克曼</td></tr><tr><td>涌入流式细胞仪</td><td>贝克曼</td></tr><tr><td>管肩</td><td>厂商VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

鉴定心肌细胞的细胞核已在组织切片挑战，因为大多数的战略仅依靠细胞质标记蛋白1。在罕见的事件，如心肌细胞增殖和凋亡需要的心肌细胞的细胞核的准确识别，分析细胞在病理条件下的稳态和重建。在这里，我们提供了一种方法来隔离验尸组织密度沉淀和免疫标记抗体对pericentriolar材料（PCM-1）和随后的流式细胞仪分选心肌细胞核。这一战略使一个高吞吐量的分析与新鲜组织和冰冻的档案材料，同样的工作的优势和隔离。这使人们有可能已经在生物银行收集研究材料。这项技术是适用和广泛的物种进行测试，适用于多种下游应用，如碳14约会3，细胞CYCLE分析，可视化胸腺嘧啶核苷类似物（如BrdU和IDU）4，转录组和表观遗传分析。

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

心肌细胞核通过密度沉淀分离与1 pericentriolar材料（PCM-1）识别和排序流式细胞仪检测心肌细胞核抗体immunolabeled。

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Research

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从部分Pancreatectomized患者的人类胰岛的分离

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

科研

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从成年小鼠分离心膜肌细胞

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

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光遗传学调节心细胞活性的机电评估

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Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

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从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

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制备用于心肌梗死后成年小鼠心脏单细胞RNA测序的非心肌细胞悬液

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...