这项工作是由DMH和TDM（NHMRC项目赠款）的赠款。 DMH是Grimwade研究员，由Miegunyah信托基金资助。

1。细胞的制备与ReAsH / FLASH标签<ol><li>使用标准的细胞培养方法，为您感兴趣的细胞株，准备直接在一个活细胞成像幻灯片的转染贴壁细胞培养。 </li><li>转染质粒TC利息根据您所选择的转染法标记的基因。 </li></ol>注意：重​​要的是使用阳性和阴性对照，以评估特异性结合的程度，TC的标签和通道之间的荧光bleedthrough评估时，收集共焦显微。因此，两种颜色（如Flash /樱桃或ReAsH / CFP或ReAsH / GFP组合），确保样品为单一颜色（如荧光蛋白单独或如果可能的话一个TC -标记蛋白结合FLASH / ReAsH但没有荧光准备蛋白质） <ol start="3"><li>转染后一到两天，轻轻300μL预热（37℃）的HBSS冲洗细胞。 </li><li>轻轻浸入1微米闪存（或ReAsH）在300μL预热的HBSS和10μM1,2 - ethanedithiol（EDT）细胞。 </li></ol>添加东部时间之前，先加入FLASH / ReAsH和缓冲区前将它添加到的细胞，它是重要的。整整30分钟的孵育37 °组织彗星文化的孵化器。在我们的经验，潜伏期较长时间显着增加的背景荧光。新的结构也应优化标签的时间和闪存/ ReAsH浓度（0.5-2μM）。 <ol start="5"><li>轻轻吸出，然后更换标签解决方案，从细胞，用300μL预热的HBSS + 250微米2,3 - dimercaptopropanol（BAL）为15分钟，在37 ° C。 </li><li>温柔的愿望，取出洗净的解决方案，并取代用300μL预热的HBSS。 </li></ol>在此之后洗净，可固定细胞多聚甲醛（3.2％溶液15分钟），虽然我们已经发现，这会增加非特异性biarsenical染料的荧光。因此，我们通常形象的细胞住在室温下。 （注，固定细胞前标签防止biarsenical染料结合。） 2。共聚焦显微镜成像的细胞<ol><li>在共聚焦显微镜，成立成像（见表1）单个荧光团的参数，并确保有之间的通道可以忽略不计bleedthrough。这样就可以实现通过检查单个荧光团（对照样品），对每个不同的荧光的荧光收购设置。调整发射波长范围，可有助于减少bleedthrough（虽然这也可以减少信号/噪声）。 </li><li>还可以调整设置，使样品中的最大荧光不饱和探测器的光电倍增管。 （这可以使用的Q - LUT的设置上SP2徕卡共聚焦检测）。一旦确定了最佳的成像设置，不改变他们之间的任何样品。 </li><li>我们通常使用的其他设置（虽然这可以优化）是一个200赫兹的扫描速度，并收集4号线的平均值和一个针孔的直径1艾里单位。如果信号/噪音是一个问题，不过，这可能导致一些损失的成像，针孔的直径可扩展。 </li><li>如果可能的话，收集12位（或更高）格式的共聚焦图像。 12位格式捕捉的每个像素点​​的亮度值（0-4095）的动态范围大于8位（0-255）。这一点很重要，以确保最富有的数据集的记录，从而最大程度的定量分析数据的质量。 </li><li>收集荧光蛋白通道（樱桃，GFP或CFP），也为biarsenical染料通道对所有样品（闪存或ReAsH）的图像。 </li></ol> 3。数据分析<ol><li> 第八您的计算机上安装软件ImageJ。 <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> </li><li>确保您的ImageJ版本有以下插件： <ul><li> <a href="http://rsbweb.nih.gov/ij/plugins/file-opener.html">http://rsbweb.nih.gov/ij/plugins/file-opener.html</a> </li><li> <a href="http://rsbweb.nih.gov/ij/plugins/image_correlator.html">http://rsbweb.nih.gov/ij/plugins/image_correlator.html</a> </li><li> <a href="http://rsbweb.nih.gov/ij/plugins/lut-editor.html">http://rsbweb.nih.gov/ij/plugins/lut-editor.html</a> </li></ul></li><li>打开ImageJ，如果使用比v1.32c旧版本，请点击下列选项，使多个图像在同一时间（可以通过按住控制按钮，当你点击不同的文件）打开： <img src="/files/ftp_upload/2857/2857step1.jpg" alt="图1" /></li><li>打开ImageJ感兴趣的图像。 ImageJ将自动定义的最高和最低分别为每幅图像的屏幕上显示的像素强度。 由于不同的图像将有different像素的强度，这意味着作为观看，显示的图像不具有可比性。 </li><li>为了确保打开的图像上同等规模的，您可以物理定义上，较低的像素强度来看待（这是不会改变图像的实际数据内容将在其他一些软件的情况下）。这些值可以设置在以下菜单： <img src="/files/ftp_upload/2857/2857step2.jpg" alt="图2" /></li><li>另一种方法是栈，汇集成一个文件，把所有的图像，并会自动调整（只供观看）在栈中的所有图像同等规模的工作。工作与数据最简单的方法是把每个通道堆栈。因此，转换荧光蛋白通道的所有协议栈，如图所示。您可以轻松地选择一个通道，通过堆叠的所有图像标题中含有一个共同的名字（例如，“CH00”）。 <img src="/files/ftp_upload/2857/2857step3.jpg" alt="图3" /></li><li>现在所有打开的图像转换为8位，以供分析。这实际上是将重新调整成一个0-255范围内（定义8位）的观看范围。 要点 ：不要超过原来的12位（或更高）格式保存，不然就会失去信息内容。请注意，一些软​​件只能显示8位的图像，所以这种格式用于数字等 <img src="/files/ftp_upload/2857/2857step4.jpg" alt="图4" /></li><li>所谓“8位转换”选项“另存为...”一个新的文件夹中保存的副本。 </li><li>检查biarsenical染料结合在一个整体形象的程度的一种方法是执行一个像素强度的相关情节。这地块每一个通道，相对于在第二个通道的相应像素值的像素值。因此一个像素高CFP荧光的位置也将在ReAsH荧光，如果有高结合。 </li><li>分析像素的合作相关，确保8位ReAsH和蔚蓝/ ​​GFP图像ImageJ开放。 </li><li>打开“图像相关”插件，如下所示。选择作为ReAsH或Flash栈和“较受欢迎”的荧光蛋白栈“我们无法找到您所查找的内容”。 <img src="/files/ftp_upload/2857/2857step5.jpg" alt="图5" /></li><li>导致堆栈可能不会显示任何详细信息 - 这是正常的。堆栈保存在一个新的文件夹称为“散点图”，并给它作为样品的相同的文件名，它是指（如“樱桃FLASH相关情节”） </li><li>要查看数据有意义，你可以做以下两个选项之一。首先转换数据，以便它在日志格式。然后以可视重新调整数据如下： <img src="/files/ftp_upload/2857/2857step6.jpg" alt="图6" /></li><li>或者，您可以直观地重新调整的数据，仅显示较低的值（如255），并显示数据，使用一个特殊的LUT。LUT（查找表）表示，被分配在图像的每个像素值的颜色表这可以用来定义一个图像的伪彩色计划是用于定义图像中的某些功能。要创建一个自定义的LUT单击“LUT的编辑器”，使一个如所示（这可以被保存并用于以后）一个新的。 <img src="/files/ftp_upload/2857/2857step7.jpg" alt="图7" /></li><li>设置范围从0-255的亮度/对比度（如上述步骤13所述）。 “锁定”在屏幕上显示的图像，图像可以转换为RGB格式，从而节省了为每个像素的颜色，红色，绿色和蓝色的8位值。 <img src="/files/ftp_upload/2857/2857step8.jpg" alt="图8" /> 复制并粘贴到其他程序的图像，首先开放的8位或16位图像。由于在第14步以上所述，指定图像的LUT的配色方案。对CFP，分配如下所述的“青色”的LUT ... <img src="/files/ftp_upload/2857/2857step9.jpg" alt="图9" /></li><li>选择图像，并把它复制到剪贴板，选择... <img src="/files/ftp_upload/2857/2857step10.jpg" /></li></ol> 4。代表性的成果： biarsenical染料标记细胞的成功是依赖于几个关键参数。首先，标签与染料的时间是至关重要的。我们发现，长时间的标签（30分钟以上），在一个高层次的非特异性背景染色的结果。图1显示了一个典型的结果为野生型亨廷顿片段的形式融合到CFP衍生蔚蓝，包含一个TC标签， 描述以前7（25Q ）。这个范例是染色与ReAsH 30分钟，并有最小的背景在缺乏TC标记的样本。我们发现，固定与多聚甲醛增加背景的细胞，而用甲醇固定废除荧光蛋白标记的荧光。因此，在可能的情况下，我们的形象细胞活。重要的是还注意到，固定前biarsenical染料标记，防止它们的结合，可能是因为训练班图案的修改。 一致的结果的另一个关键因素是细胞的密度。我们发现它的关键松散地分布，还广泛聚集可以导致不平衡，在不同的细胞biarsenical染料染色的细胞图像。 <img src="/files/ftp_upload/2857/2857fig1.jpg" alt="图1" /> 图1。Tetracysteine ​​标签和ReAsH在亨廷顿（exon1 - 25Q）蔚蓝的融合与转染活细胞染色。 TC标记是位于亨廷顿的蔚蓝融合的交界处（7） 。像素强度的相关情节，使评估ReAsH整个细胞具有约束力的差异，可以用来映射ReAsH的变化结合构象变化或配体的相互作用。

<ol>
	<li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Ann. Rev. Biochem. 67, 509-544 (1998).</li><li>Griffin, B. A., Adams, S. R., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+covalent+labeling+of+recombinant+protein+molecules+inside+live+cells.">Specific covalent labeling of recombinant protein molecules inside live cells.</a> Science. 281, 269-2672 (1998).</li><li>Uttamapinant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorophore+ligase+for+site-specific+protein+labeling+inside+living+cells.">A fluorophore ligase for site-specific protein labeling inside living cells.</a> Proc. Natl. Acad. Sci. USA. 107, 10914-10919 (2010).</li><li>Ignatova, Z., Gierasch, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein+stability+and+aggregation+in+vivo+by+real-time+fluorescent+labeling.">Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.</a> Proc. Natl. Acad. Sci. USA. 101, 523-528 (2004).</li><li>Coleman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+detection+of+prion+protein+with+biarsenical+labeling+and+FlAsH+fluorescence.">Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence.</a> Biochem. Biophys. Res. Commun. 380, 564-568 (2009).</li><li>Luedtke, N. W., Dexter, R. J., Fried, D. B., Schepartz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+polypeptide+and+protein+domain+conformation+and+association+with+FlASH+and+ReAsH.">Surveying polypeptide and protein domain conformation and association with FlASH and ReAsH.</a> Nat. Chem. Biol. 3, 779-784 (2007).</li><li>Ramdzan, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+sensors+that+distinguish+monomeric+proteins+from+oligomers+in+live+cells.">Conformation sensors that distinguish monomeric proteins from oligomers in live cells.</a> Chem. Biol. 17, 371-379 (2010).</li><li>Abramoff, M. a. g. e. l. h. a. e. s., PJ, S. J. R. a. m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Hearps, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biarsenical+dye+Lumio+exhibits+a+reduced+ability+to+specifically+detect+tetracysteine-containing+proteins+within+live+cells.">The biarsenical dye Lumio exhibits a reduced ability to specifically detect tetracysteine-containing proteins within live cells.</a> J. Fluor. 17, 593-597 (2007).</li><li>Adams, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+biarsenical+ligands+and+tetracysteine+motifs+for+protein+labeling+in+vitro+and+in+vivo:+Synthesis+and+biological+applications.">New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications.</a> J. Am. Chem. Soc. 124, 6063-6076 (2002).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Meth. 2, 905-909 (2005).</li></ol>

没有利益冲突的声明。

一种荧光蛋白和第二个染料的构象属性的方法来标记蛋白定位提供了潜在的映射不同的构象的蛋白质在细胞和改变蛋白质构象的动态的事件累积。 ReAsH / FLASH首次用于哺乳动物细胞视黄酸结合蛋白， 我 4折叠的蛋白质在细胞传感器作为。在这个例子中，绑定到一个TC标签的FLASH设计到细胞视黄酸结合蛋白，我减少了在折叠的形式展开的形式相对荧光量子产率，可以在大肠杆菌中进行?...

<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> 8以及μ-幻灯片</td><td> Ibidi </td><td> 80826 </td><td>我们发现这些玻片培养细胞成像特别有用。 </td></tr><tr><td> TC - FLASH II在细胞Tetracysteine​​标签检测试剂盒*绿色荧光*为活细胞成像</td><td> Invitrogen公司</td><td> T34561（FLASH）或T34562（ReAsH） </td><td></td></tr><tr><td>汉克斯“平衡盐溶液</td><td> Invitrogen公司</td><td> 14175-103 </td><td></td></tr><tr><td> 2,3 - 二巯基-1 - 丙醇</td><td> Sigma - Aldrich公司</td><td> D1129 - 5ML </td><td></td></tr><tr><td> 1,2 - Ethanedithiol </td><td> Sigma - Aldrich公司</td><td> 02390 - 25ML </td><td></td></tr></tbody></table>

reash/flash labeling and image analysis of tetracysteine sensor proteins in cells

荧光蛋白染料是蛋白质贩运，本地化和细胞的功能研究的重要工具。虽然荧光蛋白，如绿色荧光蛋白（GFP）已被广泛应用于蛋白质作为融合伙伴跟踪与较小的标签启用新功能的蛋白质，在构象改变，如细 ​​胞研究的利息1蛋白的性质，最近的事态发展和蛋白质协会2，3。一个小标签系统涉及tetracysteine ​​的主题（CCXXCC）基因插入到目标蛋白质，其中结合biarsenical染料，ReAsH（红色荧光）和Flash（绿色荧光），特异性高，即使是在2活细胞。 TC / biarsenical染料系统提供宿主蛋白的荧光蛋白比，这使一些新的方法来衡量的构象变化和蛋白质的相互作用4-7远不如立体的约束。我们最近开发的传感器齐聚细胞表达突变亨廷顿在一个新颖的TC标签应用程序发生突变时，聚集在神经元在亨廷顿病7。亨廷顿是两个荧光染料，荧光蛋白跟踪蛋白的位置，第二一个TC标签，它仅在单体结合biarsenical染料标记。因此，蛋白质和biarsenical染料反应之间的共定位的变化，使细胞内的空间映射微观低聚物含量。在这里，我们描述如何标注训练班标签蛋白融合在生活的哺乳动物细胞的FLASH或ReAsH和如何量化两色荧光（樱桃/ FLASH，CFP / Flash或GFP /荧光蛋白（樱桃，GFP或CFP） ReAsH组合）。

Watch this Scientific Journal Video about ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells at JoVE.com

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

biarsenical染料Flash和ReAsH特异性结合蛋白质tetracysteine​​图案，并在活细胞中的蛋白质可以选择性地标签。最近这个标签的战略已被用于开发传感器用于不同的蛋白质的构象或低聚国家。我们描述了标签的方式和方法，定量分析具有约束力。

ReAsH / FLASH标签和图像分析Tetracysteine​​传感器蛋白在细胞中

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

reashflash-labeling-image-analysis-tetracysteine-sensor-proteins

Research

JoVE Journal

Biology

16.9K 次观看.  Bio21 Molecular Science and Biotechnology Institute. biarsenical染料Flash和ReAsH特异性结合蛋白质tetracysteine​​图案，并在活细胞中的蛋白质可以选择性地标签。最近这个标签的战略已被用于开发传感器用于不同的蛋白质的构象或低聚国家。我们描述了标签的方式和方法，定量分析具有约束力。

视频: ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

科研

生物学

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

动态钙的自动分析<sup&gt; 2 +</sup&gt;在图像序列信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

测定法错误折叠的蛋白质在细胞中的降解

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

ReAsH / FLASH标签和图像分析Tetracysteine​​传感器蛋白在细胞中

ReAsH / FLASH标签和图像分析Tetracysteine传感器蛋白在细胞中