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（第二热水器）使用一个单步拉方案（第1步设置）。请务必放置在中心的每一个毛细血管加热元件，使这两个导致针头长度是一个类似的。 </li><li>拉几个毛细血管（约2为每个感兴趣的蛋白质的毛细血管）。 </li><li>商店针在一个容器中，同时确保不损坏针尖。在容器内，可用于材料，如泡沫或Blu - TACK举行显微注射​​针。 </li></ol> 2。注射蛋白质混合物的制备<ol><li>准备了10倍的显微注射缓冲液pH值在7.4 1米氯化钾和0.1 M磷酸钾（KPI），缓冲区（K 2 HPO 4和 KH 2 PO 4摩尔混合物）。这个缓冲区，可以在室温下长期存放。 </li><li>为了形象化注射，这样的荧光染料罗丹明-葡聚糖需要加以补充。在水中稀释the10x显微注射缓冲和罗丹明-葡聚糖粉末溶于5倍的显微注射缓冲液中含有20-40毫克/毫升罗丹明右旋糖酐。 </li><li>商店这个解决方案，在黑暗中，在4 ° C。它应为多达100个个人的蛋白质溶液的准备工作有足够。其它染料，如异硫氰酸荧光素葡聚糖，可以替代。 </li><li>准备溶于水纯化细胞色素 c的浓度为20毫克/毫升细胞色素 c的股票。商店细胞色素 c在-80 ° C下长期贮存和小（〜10μL）分装储存在细胞色素 c，避免反复冻融。 </li><li>准备注射10μL蛋白质混合物2μL5倍显微注射缓冲液3μL水和5μL的细胞色素 C终浓度为10 mg / mL的细胞色素 c在1x缓冲液（氯化钾100毫米，10毫米KP罗丹明-葡聚糖结合我 4-8毫克/毫升罗丹明葡聚糖）。 </li></ol> 3。细胞质显微注射细胞色素 c <ol><li>之前，以显微注射，离心16,000克的细胞色素 C和显微注射缓冲的混合物为10分钟，在4 ° C至单独的任何微粒物质可能堵塞显微注射针。 </li><li>在离心过程中，微量允许气压建立。 </li><li>放在显微镜阶段的中心一碟的细胞和细胞的重点。为了尽量减少时间，细胞外的孵化器，的数量，细胞通常返回在30分钟内的孵化器。 </li><li>吸取0.5-1微升到钝端的毛细管顶面的蛋白质混合物。小心不要吸管已离心管底部的粒子。一分钟内，蛋白质混合物将分发到针尖通过毛细作用。 </li><li>毛细管持有人的显微和位置，因此，其尖端通过在显微镜透射光经过约45 °角的针针连接牢固。 </li><li>调整针尖的位置，使其位于视野中心直接。中心针，用显微针移动，同时通过显微镜目镜。针的阴影应该是可见的。使只看到它的身影在视野的一半，表明针尖以上的细胞为中心，调整针。 </li><li>设置的微量连续流动模式，并设定工作压力为20 - 100百帕。每一针，可能需要不同的工作压力，工作压力可能会需要在显微注射过程中的调整，以保持一个稳定的的流量。 </li><li>下针，使用粗旋钮位置正上方的细胞。要做到这一点，提高显微镜的焦平面位置正上方的细胞。然后重点是降低对使用粗旋钮细胞的针，直到针尖。 </li><li>中心视野范围内的针尖，并增加通过改变显微镜物镜的放大倍率。 </li><li>慢慢放下针使用罚款操纵旋钮unti升针略高于细胞焦平面。 </li><li>检查红色荧光的罗丹明蛋白质混合物的流动。蛋白质混合物应退出针薄，络绎不绝。针应更换和重新加载如果针被攻破或如果流量是过于强烈。有时，是封闭的玻璃毛细管尖端和无流针。如果发生这种情况，更换针头或仔细降低到培养皿的底部针尖轻轻破裂。 </li><li>针尖，这是对一个细胞在大约45 °角指向的位置。然后用一个平滑的运动，降低针，而走向细胞。与第二个平滑的运动，立即扭转针的方向，从细胞中移除。 </li><li>一个成功的注入细胞往往会略微膨胀，可以通过可视化细胞内的红色荧光的罗丹明确认。有时，将意外注入细胞中的细胞核，这将是可见的的。 </li><li>继续注资，通过调整显微镜阶段，直到约50-100细胞注入细胞。当移动显微镜阶段，一定要提高显微注射针，以便它清除细胞的顶部。 </li></ol> 4。代表性的成果： 胞浆内显微注射细胞色素 C从线粒体细胞凋亡过程中模仿其释放。因此，正如所料，成纤维细胞迅速进行胞浆内显微注射的牛细胞色素 c（图1A）后，细胞凋亡。为了确保单独注射过程不负责细胞死亡，注射酵母细胞色素 c作为一个重要的控制的，因为酵母细胞色素 c是无法激活caspases 的 12 。 有趣的是，有丝分裂后的交感神经元胞浆细胞色素c（ 图 1B）8,13显着的抗。我们的实验室已经确定了内源性caspase抑制剂XIAP的是一个 14神经细胞caspase活化的关键抑制剂。因此，神经元细胞色素C注射液后死亡，XIAP的必须先成为灭活。例如，显微注射到XIAP - / -交感神经元细胞色素 C是足以让在这些细胞中caspase活化和细胞凋亡（图2）。 <img src="/files/ftp_upload/2773/2773fig1.jpg" alt="图1"/> 图1。细胞质显微注射细胞色素 C诱导成纤维细胞迅速死亡，但没有神经元。一）野生型MEFs或（B）出生后5野生型交感神经元牛细胞色素C（10毫克/毫升），连同罗丹明-葡聚糖标记注射细胞显微注射。图像显示细胞后立即注射（0小时）相同的领域，或在指定的时间。箭头指示注入细胞。比例尺，20微米。 <img src="/files/ftp_upload/2773/2773fig2.jpg" alt="图2"/> 图2。 XIAP的缺陷神经元容易胞浆细胞色素 C显微注射。产后第5天XIAP的基因敲除小鼠的交感神经元，与牛的细胞色素 C（10毫克/毫升），连同罗丹明-葡聚糖标记注射细胞显微注射。图像显示，同一领域的细胞立即注射后（0小时），或细胞色素 c显微注射后5小时（5小时）。比例尺，20微米。

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所有的动物实验程序批准的机构动物护理和使用委员会在北卡罗莱纳大学。

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 ...

activation-of-apoptosis-by-cytoplasmic-microinjection-of-cytochrome-c

Research

JoVE Journal

Biology

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

17.3K 次观看.  University of North Carolina. 在这个协议中，我们描述直接胞浆内显微注射细胞色素 C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 C蛋白进入细胞的细胞质和模仿释放细胞色素 C从线粒体，细胞凋亡过程中发生。

视频: Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

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

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmune disease, clearance of infectious agents, and the development of artheriosclerotic plaques.   In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.  Central to these studies is CD1d - the antigen presenting molecule that presents glycolipids to NKT cells.  The advent of CD1d tetramer technology, a technique developed by the Kronenberg lab, is critical for the sorting and identification of subsets of specific glycolipid-reactive T cells.  Mitch explains how glycolipid agonists are being used as therapeutic agents to activate NKT cells in cancer patients and how CD1d tetramers can be used to assess the state of the NKT cell population in vivo following glycolipid agonist therapy.  Current status of ongoing clinical trials using these agonists are discussed as well as Mitch's prediction for areas in the field of immunology that will have emerging importance in the near future.

Natural Killer T cells (NKT) are critical determinants of the immune response to cancer, regulation of autioimmunity, clearance of infection, and the development of artheriosclerotic plaques. In this interview, Mitch Kronenberg discusses his laboratory's efforts to understand the mechanism through which NKT cells are activated by glycolipid antigens.

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

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

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

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

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

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 generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

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

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

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

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

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

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

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

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

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

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

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)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

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

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

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 in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

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