Name: activation of apoptosis by cytoplasmic microinjection of cytochrome c
Uploaded: 2011-06-29T13:00:00
Description: Watch this Scientific Journal Video about Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c at JoVE.com

这项工作是由美国国立卫生研究院授予NS042197到MD支持。 AJK支持补助T32GM008719和F30NS068006。

1。生产的微量注射针<ol><li>商业预制的显微注射针（如Eppendorf公司Femtotips）是有用的，如果没有进行大量的microinjections。然而，对于那些希望建立长期的能力，为显微注射，另一种方法是在实验室采用薄壁高硼硅玻璃毛细管和商业针拉马生产的微量注射针。这也使针的形状是多种多样的，可用于不同类型的细胞。 </li><li> Narishige PC - 10显微注射针拖轮，将所有4个重量和58.0相对热定型（第?...

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	<li>Danial, N. N., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death:+critical+control+points.">Cell death: critical control points.</a> Cell. 116, 205-219 (2004).</li><li>Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+role+of+mitochondria+in+apoptosis.">The expanding role of mitochondria in apoptosis.</a> Genes Dev. 15, 2922-2933 (2001).</li><li>Hengartner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+apoptosis.">The biochemistry of apoptosis.</a> Nature. 407, 770-776 (2000).</li><li>Fuentes-Prior, P., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+structures+that+shape+caspase+activity,+specificity,+activation+and+inhibition.">The protein structures that shape caspase activity, specificity, activation and inhibition.</a> Biochem. J. 384, 201-232 (2004).</li><li>Tait, S. W., Green, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+cell+death:+outer+membrane+permeabilization+and+beyond.">Mitochondria and cell death: outer membrane permeabilization and beyond.</a> Nat Rev Mol Cell Biol. 11, 621-632 (2010).</li><li>Brustugun, O. T., Fladmark, K. E., Doskeland, S. O., Orrenius, S., Zhivotovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+induced+by+microinjection+of+cytochrome+c+is+caspase-dependent+and+is+inhibited+by+Bcl-2.">Apoptosis induced by microinjection of cytochrome c is caspase-dependent and is inhibited by Bcl-2.</a> Cell Death Differ. 5, 660-668 (1998).</li><li>Li, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+induction+of+apoptosis+by+microinjection+of+cytochrome+c.+Bcl-XL+has+activity+independent+of+cytochrome+c+release.">Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-XL has activity independent of cytochrome c release.</a> J. Biol. Chem. 272, 30299-30305 (1997).</li><li>Deshmukh, M., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+novel+event+during+neuronal+death:+development+of+competence-to-die+in+response+to+cytoplasmic+cytochrome+c.">Evidence of a novel event during neuronal death: development of competence-to-die in response to cytoplasmic cytochrome c.</a> Neuron. 21, 695-705 (1998).</li><li>Vaughn, A. E., Deshmukh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+metabolism+inhibits+apoptosis+in+neurons+and+cancer+cells+by+redox+inactivation+of+cytochrome+c.">Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c.</a> Nat. Cell Biol. 10, 1477-1483 (2008).</li><li>Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+apoptosis+by+Bcl-2:+release+of+cytochrome+c+from+mitochondria+blocked.">Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.</a> Science. 275, 1129-1132 (1997).</li><li>Gonzales, D. H., Neupert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+mitochondrial+c-type+cytochromes.">Biogenesis of mitochondrial c-type cytochromes.</a> J Bioenerg Biomembr. 22, 753-768 (1990).</li><li>Ellerby, 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=Establishment+of+a+cell-free+system+of+neuronal+apoptosis+-+comparison+of+premitochondrial,+mitochondrial,+and+postmitochondrial+phases.">Establishment of a cell-free system of neuronal apoptosis - comparison of premitochondrial, mitochondrial, and postmitochondrial phases.</a> J. Neurosci. 17, 6165-6178 (1997).</li><li>Neame, S. J., Rubin, L. L., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+cytochrome+c+activity+within+intact+neurons+inhibits+apoptosis.">Blocking cytochrome c activity within intact neurons inhibits apoptosis.</a> J. Cell Biol. 142, 1583-1593 (1998).</li><li>Potts, P. R., Singh, S., Knezek, M., Thompson, C. B., Deshmukh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+function+of+endogenous+XIAP+in+regulating+caspase+activation+during+sympathetic+neuronal+apoptosis.">Critical function of endogenous XIAP in regulating caspase activation during sympathetic neuronal apoptosis.</a> J. Cell Biol. 163, 789-799 (2003).</li><li>Estus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+gene+expression+in+neurons+during+programmed+cell+death:+identification+of+c-jun+as+necessary+for+neuronal+apoptosis.">Altered gene expression in neurons during programmed cell death: identification of c-jun as necessary for neuronal apoptosis.</a> J. Cell Biol. 127, 1717-1727 (1994).</li></ol>

直接进入细胞胞浆内显微注射细胞色素 C是一种独特而强大的的工具，它允许后线粒体调控细胞凋亡的研究。更重要的是，这种技术可以让没有使用导致细胞或线粒体损伤的代理的直接激活线粒体凋亡下游。 虽然这个协议对微量注射细胞色素 C对细胞凋亡的研究重点，这里显示的蛋白质显微注射法的一般原则也可以用于其他感兴趣的蛋白质。例如，一些研究者已?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>马克IRE2倒置显微镜</td><td>徕卡</td><td></td><td></td></tr><tr><td> PC - 10显微注射针拖轮</td><td> Narishige </td><td></td><td></td></tr><tr><td>最低工资的202微操作机器人</td><td> Narishige </td><td></td><td></td></tr><tr><td> FemtoJet微量</td><td> Eppendorf公司</td><td></td><td></td></tr><tr><td>薄壁Boroscilicate毛细管玻璃与微丝</td><td>上午系统</td><td> 615000 </td><td> 4英寸长，1.00毫米外径，内径0.75 </td></tr><tr><td>罗丹明B异硫氰酸-葡聚糖</td><td> Sigma - Aldrich公司</td><td> R9379 </td><td>平均分子量约70000大</td></tr><tr><td>牛细胞色素C蛋白</td><td> Sigma - Aldrich公司</td><td> C3131 </td><td></td></tr></tbody></table>

activation of apoptosis by cytoplasmic microinjection of cytochrome c

凋亡或程序性细胞死亡，是一个保守的和高度管制的途径，其中的细胞死亡 ， 1。可以触发细胞凋亡的细胞时遇到一个广泛的细胞毒性强调。这些侮辱启动信号级联反应，最终导致细胞色素 C从线粒体intermembrane空间释放到细胞质2。细胞色素 C从线粒体释放，是一个关键的事件触发迅速激活caspases的，关键的细胞蛋白酶，其最终执行细胞死亡3-4。 细胞凋亡通路是调节细胞色素 C从线粒体释放5点的上游和下游。为了研究线粒体调节caspase活化后，许多研究者纷纷转向直接holocytochrome C（血红素附后）蛋白质进入细胞6-9的胞浆内显微注射。细胞色素 c通常是本地化的线粒体一个血红素组的附件是必要的，使其能够激活细胞凋亡 10-11 。因此，直接激活caspases的，这是必要的，因为细胞色素 C的基因结构表达，而将导致线粒体定位和血红素附件，它将会从胞质caspases的螯合注入holocytochrome C蛋白，而不是其cDNA。因此，直接附加纯化的血红素细胞色素C蛋白的胞浆内显微注射是一个有用的工具，以模仿不使用造成细胞线粒体损伤的有毒侮辱的线粒体细胞色素C释放和凋亡。 在这篇文章中，我们描述了细胞色素 c蛋白进入细胞的显微注射的方法，用小鼠胚胎成纤维细胞（MEFs）和主要交感神经元作为例子。虽然这个协议对细胞色素 C注射液对细胞凋亡的调查重点，这里显示的技术也可以很容易地适应注射其他感兴趣的蛋白质。

Watch this Scientific Journal Video about Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c at JoVE.com

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

在这个协议中，我们描述直接胞浆内显微注射细胞色素<em&gt; C</em&gt;蛋白的成纤维母细胞和初级交感神经元。这种技术可以引进的细胞色素<em&gt; C</em&gt;蛋白进入细胞的细胞质和模仿释放细胞色素<em&gt; C</em&gt;从线粒体，细胞凋亡过程中发生。

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Research

JoVE Journal

Biology

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Interview: Glycolipid Antigen Presentation by CD1d and the Therapeutic Potential of NKT cell Activation

专访：糖脂抗原提呈CD1d及NKT细胞活化治疗潜力

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious ...

科研

生物学

Generation of Stable Transgenic C. elegans Using Microinjection

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Microinjection of Xenopus Laevis Oocytes

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Synthesis of an In vivo MRI-detectable Apoptosis Probe

合成的<em&gt;在体内</em&gt; MRI检测细胞凋亡的探讨

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...