Name: use of lysotracker to detect programmed cell death in embryos and differentiating embryonic stem cells
Uploaded: 2012-10-11T13:00:00
Description: Watch this Scientific Journal Video about Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells Video at JoVE.com

我们感谢帮助编辑的马里亚尼实验室的协议。这项工作是由一个摆围博士后培训津贴（JLF），的CIRM的BRIDGES实习（TZTT），罗伯特·E.五月R.莱特基金会（FVM），美国南加州大学（FVM）。

1。 LysoTracker染小鼠胚胎<ol><li>生成的小鼠胚胎放置年轻女性到男性螺柱笼（5-6周龄）。监视以下早晨指示，交配阴道栓的外观发生。如果女性怀孕了，中午在这一天被称为0.5 DPC（天交配后）。这里介绍的是该程序的胚胎7-13 DPC的理想选择。 </li><li>在胚胎的利益，根据经批准的协议和安乐死的女性，切除子宫。从一个10厘米的培养皿中，用Hanks，BSS（无酚红）的蜕膜中取出胚胎。删除多余的胚胎?...

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细胞凋亡的重要标志包括与TUNEL检测的DNA损伤的检测，沿与caspase活性的检测（检测的单克隆抗体或结合分子如ZVAD-芬兰马克），观察细胞的变化，和介绍的磷脂酰丝氨酸（PS ）在膜表面上（Annexin V结合）中检测到的。与每个这些检测方法也有一些缺点。例如，TUNEL法检测中使用的末端转移不穿透超越几个细胞层。膜联蛋白V可以标记细胞不进行细胞凋亡，如那些具有的膜破坏，（膜联蛋白V得到在细?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td></td><td> 目录编号 </td></tr><tr><td> DND-99 LysoTracker红， </td><td> Invitrogen公司</td><td> ＃L-7528 </td><td></td></tr><tr><td>汉克斯BSS </td><td> Invitrogen公司</td><td> 14025-076 </td><td></td></tr><tr><td>多聚甲醛</td><td> EMD </td><td> EM-PX0055-3 </td><td></td></tr><tr><td> Vectashield </td><td> VECTOR </td><td> H-1200 </td><td></td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 10-013-CV </td><td></td></tr><tr><td>非必需氨基酸</td><td> Cellgro </td><td> 25-025-CI </td><td></td></tr><tr><td>丙酮酸钠</td><td> Cellgro </td><td> 25-000-CI </td><td></td></tr><tr><td> FBS </td><td> Hyclone公司</td><td> SH30071.02 </td><td></td></tr><tr><td>笔链球菌</td><td> Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td> β-巯基乙醇，50mM的</td><td> Invitrogen公司</td><td> 21985-023 </td><td></td></tr><tr><td> LabTek-II玻片 （8孔） </td><td> Nalge Nunc公司国际</td><td> 154534 </td><td></td></tr><tr><td> 0.1％明胶</td><td>密理博</td><td> ES-006-B </td><td></td></tr><tr><td>的Dulbecco PBS（D-PBS） </td><td> Cellgro </td><td> 21-031-CV </td><td></td></tr><tr><td></td><td></td><td></td><td>解决方案食谱 4％多聚甲醛 100毫升： <ol><li>混合4克多聚甲醛，90毫升H 2 O，和NaOH（2N NaOH中的下降）。多聚甲醛不会进入溶液中，直到添加了一些NaOH溶液的pH值增加。 </li><li>搅拌并加热，在60℃，直到所有的粉末是在溶液中（约10-20分钟）。 不要过热。 </li><li>加入〜10毫升的10倍的PBS中达到最终体积为100ml。 </li><li>方便的（〜10毫升〜40毫升）的等分试样在-20℃下储存。 </li></ol> 警告：在&#39;做样子&#39;形式（压缩的小颗粒）的多聚甲醛减粉末，因此，可以测量以外的机罩。但是你还是应该戴防尘口罩（N95至少）在处理过程中。 EB文化传媒对于500毫升EB媒体： <table border="1"><tbody><tr><td> DMEM： </td><td> 404.5毫升 </td></tr><tr><td> FBS： </td><td> 75.0毫升</td></tr><tr><td> L-谷氨酰胺： </td><td> 5.0毫升</td></tr><tr><td>青霉素/链霉素： </td><td> 5.0毫升</td></tr><tr><td>非必需氨基酸： </td><td> 5.0毫升</td></tr><tr><td>丙酮酸钠： </td><td> 5.0毫升</td></tr><tr><td> β-巯基乙醇： </td><td> 500微升</td></tr></tbody></table></td></tr></tbody></table>

use of lysotracker to detect programmed cell death in embryos and differentiating embryonic stem cells

程序性细胞死亡（PCD）发生于成人，以维持正常组织稳态和胚胎发育过程中形成组织和器官1,2,6,7。在开发过程中，有毒化学物质或基因的改变可以导致PCD增加或改变PCD的模式，导致发育异常和出生缺陷3-5。要了解这些缺陷的病因学，胚胎的研究，使用区分胚胎干细胞（ES细胞） 在体外实验中，可以补充。 细胞凋亡是一种形式的PCD研究，既包括内在的和外在的信号，激活半胱天冬酶的酶级联。细胞特性的变化包括膜皱缩，核收缩，且DNA碎片。不涉及其他形式的PCD半胱天冬酶的激活，最终的结果可能是长期自噬。无论PCD的途径，死亡的细胞需要被删除。在成年人中，免疫细胞PERFORM功能，而在胚胎的免疫系统尚未开发，拆除产生的一种替代机制。这种机制涉及到邻近的细胞（称为“非专业吞噬细胞”），吞噬作用，他们认识到表面上的死细胞和吞噬8-10“吃我”的信号。吞噬后，碎片带来的溶酶体降解。因此无论PCD机制，溶酶体活性的增加可以增加细胞死亡相关。 要学习PCD，一个简单的分析，可视化厚的组织和多层区分文化的溶酶体中是有用的。 LysoTracker染料是一种高度可溶性小分子，被保持在如酸性亚细胞溶酶体11-13。的染料是采取了通过扩散和通过循环。由于是不是一个障碍，可视化的PCD在厚厚的组织和多层次的文化渗透是可能的12,13 </支持&gt;。与此相反，TUNEL（末端脱氧核苷酸转移酶缺口末端标记法）分析14，仅限于小样本，组织切片和单层培养，因为此过程需要进入/渗透终端转移。 相反，扩散和溶解的溶剂苯胺蓝，DND-99 LysoTracker红，是可以解决的，明亮的，稳定的。染色可以可视化与标准荧光或共聚焦显微镜在整个贴装或部分，使用水性或溶剂型的安装媒体12,13。在这里，我们描述了使用此染料看，PCD在正常和 Sonic Hedgehog null小鼠胚胎的协议。此外，我们表现出ES细胞培养鉴别分析PCD，并提出一个简单的量化方法。总之，LysoTracker染色检测PCD的其他方法可以是一个很好的补充。

Watch this Scientific Journal Video about Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells Video at JoVE.com

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells Video (Video) | JoVE

我们提出了一个简单的协议，可视化地区的小鼠胚胎中的细胞程序性死亡（PCD）和鉴别胚胎干（ES）细胞培养，使用高度水溶性染料LysoTracker。

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Unit 1

Cell Death

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Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction Video (Video) | JoVE

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...