作者感谢基金会Filarete提供的帮助和支持，在此文章的发表。我们还要感谢炫酷那么Europeo二Nanomedicina为使用该Tecnai G2透射电子显微镜。

1。质粒，细胞培养，转染，并与ER荧光蛋白<ol><li>在本研究中使用的质粒由GFP的增强版本融合在其C-末端的尾部区域的大鼠色素b的ER亚型（5）通过接头序列（如B（5）此处略）。尾部区域包含整个序列（Pro94-Asp134），该保持天然B的胰蛋白酶切割位（5），其中包括17个残基的TMD（跨膜结构域），由上游和下游的极性序列侧接（UPS和DPS）后膜相关。连接器包括myc表后跟[（甘氨酸）4辑] 3，并且整个的cDNA被插入到哺乳动物表达载体pcDNA3的Hind3-Xba1位点。该质粒的构造细节在以前的出版物中，它被称为GFP-ER 16已被描述。 </li><li>生长的COS-7细胞在Dulbecco改进的Eagle培养基（DMEM），补充有10％羊升牛血清，2mM L-谷氨酰胺，1％青霉素/链霉素的培养箱中，在37℃和10％的CO 2。 </li><li>转染。板3×10 5个细胞在6孔板中的圆形玻璃盖玻片，并在次日，如制造商所述转染与JETPEI系统。注意，最优JETPEI / DNA比率，以建立根据所使用的质粒和细胞系的最大转染效率进行了测试:在我们的情况下，JETPEI:2:1的DNA比导致70-80％的转染效率。 </li></ol> 2。实时荧光扫描共聚焦显微镜<ol><li>活细胞成像。把在其上的转染细胞接种到一个钢细胞培养室，用于24毫米盖玻片填充用DMEM W / O型酚红，补充有10％FBS，2mM L-谷氨酰胺，1％青霉素/链霉素，25mM的HEPES盖玻片，50微克/毫升放线菌酮和1:100 OxyFluor以防止样品从光漂白。一个SP5共焦microscopë配备有温度控制的CO 2培养箱（37℃和5％CO 2）用于活细胞成像的实验中，使用488nm的激光和一个五十分之五百二十五带通发射过滤器D的GFP-ER被可视化。 </li><li>漂白（FRAP）后荧光恢复。绘制感兴趣区（ROI）的区域，对应于OSER结构，它采用20次迭代和488 nm的组合（30毫瓦氩激光的100％，相当于5.5-6μW在样品）和405 nm的漂白（60％的一个30毫瓦二极管405的激光，对应于11.6μW在样品）激光器，在我们的经验，导致了高效率和快速漂白。 </li><li>通过采取一个单一的框架每10秒，10分钟（像素时间= 1.61微秒/像素）记录荧光的复苏漂白的投资回报。 </li><li>荧光损失漂白（FLIP）。绘制对应于一OSER结构的投资回报率，并且如上所述的漂白剂。漂白重复每30秒，和后漂白图像被记录，每10秒进行30分钟（像素时间= 1.61微秒/像素）。 </li><li> FRAP和FLIP分析。所有的图像都使用ImageJ软件（分析<a href="http://rsbweb.nih.gov/ij/download.html" target="_blank">http://rsbweb.nih.gov/ij/download.html</a> ）。在FRAP实验中，漂白ROI的荧光恢复测量随着时间的推移和归一化到漂白细胞的总荧光，它总是检查，以随时间恒定。 </li><li>对于FLIP实验中，得出一个投资回报率的漂白OSER和覆盖整个细胞外。测量其荧光强度随着时间的推移和正常化到ROI的荧光水平，以纠正在荧光任何减少所造成的成像本身上绘制一个未漂白细胞。 </li><li>在所有的实验中，从感兴趣区域的荧光强度中减去背景信号（在细胞外的区域确定）。最后，使用石墨绘制的结果aphPad Prism软件。 </li></ol> 3。超微结构分析透射电子显微镜的手段鉴于许多试剂的毒性，所有的程序都要进行穿着适当的实验室。大衣和手套在通风橱内。 <ol><li>从培养皿中取出盖玻片后，固定生长在培养皿如使用过滤的2％戊二醛的0.1M二甲胂酸盐缓冲液，pH 7.4的单层的底部，在室温下10分钟，剩余的细胞。 </li><li>采用聚四氟乙烯刮板刮去细胞，并将它们转移到1.5ml离心管中。通过离心的方法使细胞沉淀以9,000 g下10分钟。除去上清液，加入新鲜固定液，并留下过夜，4°C。 </li><li>清洗用缓冲液的颗粒，然后用1％四氧化锇在二甲胂酸盐缓冲液1小时，在室温下的溶液中后固定。 </li><li>冲洗用超纯水，以及整块污渍1％乙酸双氧铀在蒸馏水中20-60分钟之间。 </li><li>脱水的样品中增加的乙醇系列（70％，80％，90％，100％，100％，每次10分钟），并在两次环氧丙烷简要地洗涤（每次15分钟）。 </li><li>渗入样品中的氧化丙烯的Epon +（1:1）的混合物（从2小时到过夜）。 </li><li>嵌入的Epon环氧树脂固化物在60℃下至少24小时。 </li><li>部分使用超薄切片机LEICA UC6配备有45°的金刚石刀，以获得区段的厚度为60-70纳米的手动修整树脂集团。收集300目铜网上的部分。 </li><li>染色对电网的部分用醋酸铀（20分钟）的饱和溶液和柠檬酸铅（7分钟），通过浸在双蒸过滤水彻底清洗网格，并允许他们在室温下干燥。 </li><li>染色的网格使用的是TECNAI G2的透射电子显微镜观察和图像是使用博特捕获OM式CCD摄像头在不同的最终放大倍率（一般为6,000-39,000倍）。 </li></ol>

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作者什么都没有透露。

本文所描述的协议和成像方法已被用于研究转助教荧光蛋白居民在活细胞的内质网的分布和流动。我们也分析了这个亚细胞区室中通过超微结构分析装置的体系结构中的过度表达，这些蛋白质的效果。 活细胞共聚焦成像和电子显微镜的组合代表是一个非常强大的研究蛋白质的动态性能的手段，并可能提供有关蛋白质功能的重要信息。所描述的方法不是费时（一般工作三天）?...

绿色荧光蛋白（GFP）及其变种光谱和荧光显微镜的并行开发的发现，开辟了全新的途径，在细胞中蛋白质的行为进行调查。技术，如荧光漂白恢复（FRAP）和荧光损失漂白（FLIP），这是因为荧光的内在能力，以扑灭在强光照射下其荧光的可能后，基于共聚焦活细胞成像和使用的转荧光融合蛋白1-3。它们被广泛用于评估蛋白质不仅是本地化，而 ​​且他们的流动性和膜泡运输，它可以揭示有关它们的功能4重要线索。 真核细胞的独特之处在于具有特定的脂质和蛋白组成的细胞内隔室的存在。虽然细胞器是物理隔离器版，它们需要彼此通信，并以维持细胞稳态交流分子成分。分泌途径保证了在ER中合成的蛋白质和脂质到达正确的最终目标，其中它们发挥它们的功能。细胞内细胞器，也可以通过动态接触部位，使分子（脂质）向隔间之间直接交换连接。此外，许多蛋白已到组装在大异聚复合物，或与特定的脂类物质（脂质筏/微区）为了成为功能活性或者被运送到最终目的地相关联。所有这些生物方面极大地影响蛋白质的动力学性质，并因此可以通过如下所述的技术手段进行适当的调查。 我们的团队已经广泛应用于FRAP和FLIP结合电子显微镜来研究内质网的结构和它的不同的子域。内质网是分泌途径的第一站和在蛋白质和脂质排序5起关键作用。这是一个高度动态的细胞器，其独特的子域名反映其许多不同的功能（ 如蛋白质和脂质的合成和贩运，蛋白质折叠，Ca 2 +的储存和释放，以及异物代谢）。然而，尽管它们在形态上，空间上，和功能上不同，这些结构域彼此连续，并且它们的相对丰度可在细胞生理和病理条件下进行修改。最有名的和内质网中，通常在空间上分离的结构域是核膜和平滑和粗糙的ER，但是，我们和其他人已经证明，有ER结构具有更复杂的结构和三维组织在各种细胞类型和在生理条件下的组织，也可以由紧张刺激如缺氧，药物诱导的手段管理或过度表达的ER驻留跨膜蛋白2,6（以及其中的参考文献）。 我们最近还展示了这样的结构在人类疾病中1,7的细胞模型的存在。从滑面内质的堆积潴发起，他们分别获得组织光面内质网（OSER）的总称，2003年6，虽然他们也被称为karmellae，片状结晶 ，并且晶体ER他们的架构的基础上，像他们的大小，可以改变。之后，细胞转染绿色荧光蛋白融合到尾锚（TA）ER居民蛋白质（D EGFP-ER），绿色荧光蛋白在转弱二聚化倾向显着地改变了ER的组织和结构的细胞质区域。 FRAP和FLIP实验表明使得 d EGFP-ER是自由OSERs内扩散，并且它从网状ER移动到OSER 反之亦然&lt;事实/ em&gt;的指示聚集是连续的与周围的网状急诊室。超微结构分析使我们能够将荧光数据与OSER体系结构和组织在纳米尺度水平上的详细描述关联:OSERs总是由成堆的光滑ER配对囊泡的，但可能有不同形式的空间组织，如规则排列的正弦数组或轮生，或六角形"晶体"管阵列。这些重排导致立方形态8的，因为它们在细胞中发现的生理条件9和下面的压力，如缺氧10，药物治疗11和癌症9下，可有显 ​​著电位超微结构标记。 使用GFP融合蛋白第一次演示之后，我们使用了成像实验，分析ER结构域的增殖响应于药物治疗12，ASSESS荧光蛋白的倾向oligomerise在细胞13，并调查的突变，ALS联TA蛋白在ER起源的细胞内聚集，可能是有关其致病1,8的形成中的作用。它已被提出，形成的细胞内聚集物（这发生在许多神经退行性疾病14）可以设计成防止有毒的突变蛋白和周围的细胞成分15之间的相互作用的一种保护机制。 以下是对光学和电子显微镜方法考察构建体的C-末端的疏水性结构域被插入到内质网的膜，并且其动态行为的分析以及其过度表达对ER域的效应的组合的说明在培养的细胞结构（参见图1的实验方案的流程图）。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>41966029</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dulbecco's Modified Eagle medium (DMEM) w/o phenol red</td>
			<td>Invitrogen</td>
			<td>31053028</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Invitrogen</td>
			<td>10270106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen/Strep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-Glutamine 200 mM solution</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td></td>
		 </tr>
		 <tr>
			<td>jetPEI</td>
			<td>Polyplus Transfection</td>
			<td>PP10110</td>
			<td></td>
		 </tr>
		 <tr>
			<td>OxyFluor</td>
			<td>Oxyrase Inc.</td>
			<td>OF-0005</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutaraldehyde Grade I</td>
			<td>Sigma Aldrich</td>
			<td>G5882</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Cacodylate Trihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C0250</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Osmium Tetroxide 4% solution</td>
			<td>Electron Microscopy Science</td>
			<td>19150</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Uranyl Acetate dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>73943</td>
			<td>slightly radioactive</td>
		 </tr>
		 <tr>
			<td>Propylene Oxide</td>
			<td>Sigma-Aldrich</td>
			<td>82320</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EPON embedding medium kit</td>
			<td>Sigma-Aldrich</td>
			<td>45359-1EA-F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lead Citrate </td>
			<td>Electron Microscopy Science</td>
			<td>17800</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bench top centrifuge</td>
			<td>Eppendorf</td>
			<td>5415 D</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spectral Confocal Microscope </td>
			<td>Leica Microsystems</td>
			<td>TCS SP5</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CO2 Microscope Cage Incubation System </td>
			<td>OkoLab</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ultramicrotome </td>
			<td>Leica Microsystems</td>
			<td>UC6</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Diamond knife </td>
			<td>Diatome</td>
			<td>Ultra 45 &deg;</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmission Electron Microscope </td>
			<td>FEI</td>
			<td>Tecnai G2</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GraphPad Prism Software</td>
			<td>GraphPad Software, Inc</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>steel culture cell chamber for 24 mm coverslip</td>
			<td>Bioscience Tools</td>
			<td>CSC-25</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Electron Microscopy grids</td>
			<td>Electron Microscopy Science</td>
			<td>G300Cu</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

visualization of endoplasmic reticulum subdomains in cultured cells

在真核细胞中的脂质和蛋白质细胞区室之间不断交换，但这些保留其独特的组成和功能，尽管激烈的interorganelle分子的流量。在本文中描述的技术是强大的研究蛋白质和脂质的流动性和贩运体内 ，并在其生理环境的手段。光漂白（FRAP）和荧光损失在光漂白（FLIP）后荧光恢复被广泛用于活细胞成像技术通过外 - 内吞途径，细胞器或子隔离之间的连续性，形成蛋白质复合物，以及蛋白质定位研究细胞内运输所有这些脂质微区，可生理和病理条件下观察。这些方法的局限性主要是由于使用了荧光融合蛋白，以及它们的潜在缺点包括伪迹过表达离子在细胞内和在标记的和天然蛋白质的折叠和定位的差异的可能性。最后，由于光学显微镜（约200纳米）的分辨率的极限不允许急诊室或特定的小班，可以在细胞应激状态下起源的精细结构的研究（ 即缺氧，药品监督管理，过度表达的跨膜ER常驻蛋白）或病理条件下，我们结合培养的转染细胞的活细胞成像与超微结构分析了基于透射电子显微镜。

图2显示了蛋白质的流动性的一个例子FRAP研究。 ðEGFP-ER蛋白的迁移率证明了在漂白漂白OSERs后迅速荧光恢复。对于定量分析，在中场休息时和移动部分均来自实验测得的数据进行拟合以下单指数方程: F（T）= F + 后 （F REC-F后 ）（1-E-T /τ） 其中F是后荧光信号漂白后，F REC<...

Watch this Scientific Journal Video about Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells at JoVE.com

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

我们描述了使用调查转染荧光蛋白通过活细胞的共聚焦成像装置驻留在内质网（ER）的分布和流动的成像方法。我们还超微结构分析其表达对这个亚细胞区室的结构的效果。

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

visualization-of-endoplasmic-reticulum-subdomains-in-cultured-cells

Research

JoVE Journal

Biology

12.9K 次观看.  Fondazione Filarete. 我们描述了使用调查转染荧光蛋白通过活细胞的共聚焦成像装置驻留在内质网（ER）的分布和流动的成像方法。我们还超微结构分析其表达对这个亚细胞区室的结构的效果。 

视频: Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

科研

生物学

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

染色体制备培养细胞

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

细胞器运输的培养<em&gt;果蝇</em&gt;细胞:S2细胞系和原代神经元。

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice&#160;and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer&#39;s disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

中G3BP应力颗粒动力学在Live原代细胞可视化

SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

在培养的哺乳动物细胞大规模并行报告基因分析

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

成像本地CA<sup&gt; 2+</sup&gt;在培养的哺乳动物细胞信号

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the large size of the human BRCA2 protein (approximately 390 kDa). We report modifications of standard siRNA transfection and immunoblotting protocols to silence human BRCA2 and detect endogenous BRCA2 protein, respectively, in human epithelial cell lines. Key steps include a high siRNA to transfection reagent ratio and two subsequent rounds of siRNA transfection within the same experiment. Using these and other modifications to the standard protocol we consistently achieve more than 70% silencing of the human BRCA2 gene as judged by immunoblotting analysis with anti-BRCA2 antibodies. In addition, denaturation of the cell lysates at 55 &#176;C instead of the conventional 70-100 &#176;C and other technical optimizations of the immunoblotting procedure allow detection of intact BRCA2 protein even when very low amounts of starting material are available or when BRCA2 protein expression levels are very low. Efficient silencing of BRCA2 in human cells offers a valuable strategy to disrupt BRCA2 function in cells with intact BRCA2, including tumor cells, to examine new molecular pathways and cellular functions that may be affected by pathogenic BRCA2 mutations in tumors. Adaptation of this protocol for efficient silencing and analysis of other &#39;large&#39; proteins like BRCA2 should be readily achievable.

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

沉默<em&gt; BRCA2</em&gt;以确定新的BRCA2调控的生物学功能在人体细胞培养

Silencing of the tumor suppressor protein BRCA2 and its detection by conventional biochemical analyses represent a great technical challenge owing to the ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...