我们非常感谢S.泰勒提供的pLPCX组蛋白H2-GFP质粒。我们感谢R. Mertelsmann为持续的支持。这项工作是由德国研究联合会的支持。

1。显微镜玻片上播种的U2OS细胞周期蛋白B-SNAP记者细胞（克隆11个单元6） <ol><li> Trypsinize汇合的的SNAP记者被允许异步增长对数生长期至少48小时的细胞，。 </li><li>与8孔显微镜室（恒定细胞分布）。 </li></ol>用于播种的细胞到8以及显微镜室在一个恒定的分布在整个表面的显微镜室，离心10,000个细胞，并重新悬浮在350μl的无酚红的正常生长培养基（补充有10％牛胎儿血清，青霉素/链霉素和丙酮酸钠）。细胞悬浮液转移到显微镜室（ 图2）。 使用8孔显微镜室（最大细胞密度在中心）。 用于播种细胞的中心显微镜chamb的在更高的密度在呃，用300μl的无酚红的正常生长介质加载腔室。仔细添加5000细胞的显微镜室（ 图2）的中心。 工作96以及特殊的光学板（恒定细胞分布）。 在一个恒定的分布在整个表面上的阱用于播种到96孔板中的细胞，离心5000细胞，并重新悬浮于300μl的无酚红的正常生长介质中。细胞悬浮液转移到96孔板中。根据所需的井含细胞的总数，调整细胞数和总体积的悬浮液介质中（ 图2）。 工作96以及特殊的光学板（最大细胞密度在中心）。 对于细胞接种到96孔板上，小心地加入1500细胞在15μl的无酚红的正常生长介质中的小型会议室升下降到各孔中，以达到限制细胞生长的孔中（ 图2）的中心的中心。 <ol start="3"><li>允许标准细胞培养条件下（37℃，100％的空气湿度，5％CO 2）下的生长至少18小时的种子细胞。 </li></ol> 2。记者细胞染色与SNAP基<ol><li>前30分钟的染色程序的开头允许无酚红的正常生长培养基的等分试样升温至37℃。 </li><li>对于容易处理的SNAP基板的（在我们的例子TMR星级）溶解在DMSO中的TMR星级在400μM的原液，它可以储存于-20℃下以获得一个浓度</li><li>染色前，在200μl的无酚红正常的生长培养基中（37℃），得到最终的标签的浓度为1μM的0.5微升的TMR星级原液稀释。 </li><li>删除异步生长的细胞正常生长培养基从孵育在labeling介质在标准培养条件下25分钟。 </li><li>拆下标签介质和洗涤细胞4次，用无酚红的正常生长培养基中（37℃）。孵育细胞在300μl的无酚红的正常生长培养基中（37℃）30分钟。运输到显微镜之前更换培养基用新鲜的无酚红的正常生长培养基中（37℃），以除去残留的未结合的SNAP基板。 </li><li>运输细胞显微镜在发泡胶框上预先温热（37℃），以尽量减少温度变化的热块。 </li></ol> 3。荧光强度的测量<ol><li>前两小时，分析的气候室中的空气温度调节到37℃，在干燥的模式，以便使与它的所有组件的整个显微镜到所需的温度。预加热湿度设定之前，重要的是要避免缩合和随后的损坏显微镜。 </li><li>调节空气湿度吨“O 60％，CO 2至5％之前开始分析的。 </li><li>开始扫描^ R采集软件定义标准设置（ 见表1）。 </li><li>定义的位置的孔，以进行分析。 </li><li>定义采集周期时间（ΔT）的绝对数量的采集周期。 </li><li>如果需要更高的井数的分析，选择硬件自动对焦，否则就足够了单独使用软件的自动对焦。 </li><li>开始的前两个采样周期的采集和监督。该显微镜将专注于组蛋白的H2-GFP信号，随后在该通道的第一图像采集前的过滤器被改变，并获取相应的TMR星图像（ 图3）。然后重复内的阱和要检查的每一个的孔的所有位置，再次重复之前的下一个周期。 </li></ol> 4。分析蛋白水解型材U唱扫描^ R <ol><li>启动扫描^ R分析软件和分析图像的细胞核由组蛋白H2-GFP，定义为主要对象，可视化，使用的阈值，基于信号强度和在分离周边小区的分水岭算法来协助。的细胞核与细胞质组成的子对象，应该建立TMRstar分析。 （主要对象的重要属性是X和Y位置，时间，最大和平均强度为主要对象的总强度除以区域TMRstar子对象的GFP）。这个分析过程可能需要几个小时，由于大量的数据数量。 </li><li>切换到跟踪模式，以可视化的子对象平均TMR星荧光强度随着时间的推移细胞的痕迹，分配给分析的主要对象（ 图4）。要检查的细胞的数目可以通过选通持续至少140次循环的这些测量值，并具有高的最大强度的H2-GFP收窄。展望在较大数量的细胞，允许第一代表和客观的视图（ 图4）。 </li><li>选择感兴趣的小区的痕迹，以可视化组蛋白的H2-GFP和TMR星级荧光同时在单细胞水平上（ 图5A）。 </li><li>使用利益的单元格上单击鼠标右键，并说明组蛋白H2-GFP和细胞周期蛋白B-SNAP的TMR星为每一个时间点产生一个导出的图片库。 </li><li>更改的人口模式的扫描^ R分析软件和门的区域利益的细胞上的X和Y散点图（ 图5B）表示。 </li><li>应用新的散点图窗口选通区域和可视化意味着TMR星荧光强度随着时间的推移（ 图5C）。 </li><li>出口到Microsoft Excel中的数据（时间和荧光强度）为进一步计算。 </li></ol> 5。代表性的成果 无花果URE 5D和图5E示出细胞周期蛋白B动力学，由TMR星级荧光强度的曲线，表示一个单元，通过一个常规的有丝分裂所得无染色体错位的迹象（ 图5E）。核膜击穿后的细胞质（NEBD，如所表示的红色三角形）压实后，TMR星荧光强度显示的同构的窗口（在图中的明亮区域）的突然增加，直到达到时，细胞进入中期，6。荧光强度保持在一个稳定的水平，只要通过前期和中期的细胞进入，然后开始迅速下降，一旦所有的染色体已建立了稳定的中期板（ 图5D和5E）。这一下降过程中染色体分离之前后期（蓝色曲线上的点）。在后期有丝分裂的的质开始decondense（蓝色条）和单元采用相间形态，而荧光强度曲线接近高原是低于高原前有丝分裂（ 图5D和5E）。 <table border="1"><tbody><tr><td> 自动对焦设置 </td><td> 组蛋白H2-GFP（主要对象获取设置） </td><td> 细胞周期蛋白B-SNAP标有TMR星 （采集设置） </td><td> 采集循环时间重复 </td></tr><tr><td>自动对焦+ / -39微米粗24层 精细自动对焦 + / -5.4微米14层</td><td> GFP过滤器设置： 曝光时间：100毫秒 光照强度：25％ </td><td> TRITC过滤器设置： 曝光时间：150毫秒 光照强度：33.3％ </td><td> 2〜5分钟。 </td></tr><tr><td> GFP过滤器设置： 曝光时间：12毫秒 光照强度：12.5％ </td><td></td><td></td><td>继续分析（限于通过减少空气湿度为60％至48小时） </td></tr></tbodY&gt; </table> 表1中。标准设置用于分析细胞周期蛋白B的蛋白质水解。组蛋白用作H2-GFP细胞周期蛋白B-SNAP荧光强度测量的参考结构来定义的焦点平面。聚焦过程中的光强相比，图像采集设置，以避免累积光毒性较低。 <img alt="图1" src="/files/ftp_upload/4239/4239fig1.jpg" /> 图1。细胞周期蛋白B蛋白水解通过表达的嵌合细胞周期蛋白B衍生物与类似的内源性蛋白的降解特性的测量示意图 。脉冲追踪标记后的荧光强度的下降是APC / C活性的措施。 <img alt="图2" src="/files/ftp_upload/4239/4239fig2.jpg" /> 图2。分析定期分配的8孔幻灯片或96孔板中的细胞。细胞的整个表面的阱是通过再悬浮于最终体积为300微升，并建议，如果只有一个或几个位置手动定义进行分析。富集的中心区域中的单元格的实现通过添加细胞的预充液或空井中心。这种技术建议的情况下在不同的井的自动图像采集。 <img alt="图3" src="/files/ftp_upload/4239/4239fig3.jpg" /> 图3。序列的数据采集 A）检测组蛋白H2-GFP用于在有丝分裂过程中染色体对准焦点平面的定义和监测。 B）的TMR星级的荧光强度的荧光强度是脉冲追踪的绝对量，标记的细胞周期蛋白B-SNAP的量度。 <img alt="图4" src="/files/ftp_upload/4239/4239fig4.jpg" /> 图4。 ^ R代表扫描分析软件为基础的描写平均TMR星荧光强度（总TMRstar强度除以面积）随着时间的推移选择的代表电池跟踪（蓝色）。相应的图像（如右图所示）允许的同步可视化，组蛋白：H2-GFP（绿色）和细胞周期蛋白B-SNAP（红色）。 <a href="http://www.jove.com/files/ftp_upload/4239/4239fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图5" src="/files/ftp_upload/4239/4239fig5.jpg" /> 图5：A + B）举例门点的单元格的XY散点图的利息。 C）使用散点图的平均TMRstar的荧光强度（的总TMRstar强度除以面积）随着时间的推移可视化。 D + E）代表同构的窗口（上图）brigher领域之间的核被膜破裂NEBD（）和：染色质decondensation（蓝色条），在Microsoft Excel中产生的荧光强度曲线，细胞周期蛋白B-SNAP。后期用T表示他的蓝点在曲线上。 <a href="http://www.jove.com/files/ftp_upload/4239/4239fig5large.jpg" target="_blank">点击此处查看大图</a> 。 动画1。 <a href="http://www.jove.com/files/ftp_upload/4239/4239movie1.avi" target="_blank">点击此处查看补充电影</a> 。

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没有利益冲突的声明。

我们在座的活细胞成像为基础的方法，促进细胞周期蛋白B同时监测蛋白质和染色体排列。这种方法允许在单细胞水平上研究的有丝分裂控制中泰然自若的细胞群。细胞周期蛋白B降解曲线的APC / C的活性和促进直接的见解，从而间接地反映了状态的SAC 6。 这种方法，即使建立了基于扫描^ R软件，可以很容易地被复制任何类似的显微镜站。此外，使用我们的逆转录病毒的?...

<table border="1"><tbody><tr><td> 产品名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>报道细胞系</td><td>产生的房子，6 </td><td>克隆11个记者细胞 （U2OS基于细胞周期蛋白B-SNAP细胞） </td><td></td></tr><tr><td>逆转录病毒的细胞周期蛋白B-SNAP表达载体</td><td>产生的房子，6 </td><td> pLNCX2细胞周期蛋白B mut5-SNAP </td><td></td></tr><tr><td>免费的phenolred-DMEM </td><td> Gibco公司</td><td> 21063-029 </td><td> FCS，丙酮酸钠，青霉素/链霉素补充要求</td></tr><tr><td> SNAP-细胞TMR明星</td><td> New England Biolabs公司</td><td> S9105S </td><td>原液400μM的在DMSO中</td></tr><tr><td>特别Opstics板，96孔</td><td>深圳科诗特</td&gt; <td> 3720 </td><td></td></tr><tr><td> μ-幻灯片8，ibiTreat </td><td> Ibidi </td><td> 80826 </td><td></td></tr><tr><td>显微镜装置</td><td>奥林巴斯</td><td> IX-81倒置显微镜与气候室</td><td></td></tr><tr><td>目标</td><td>奥林巴斯</td><td> UPLSAPO 20X物镜（NA 0.75） </td><td></td></tr><tr><td>采集软件</td><td>奥林巴斯</td><td>扫描^ R采集软件（v.2.2.09） </td><td></td></tr><tr><td>分析软件</td><td>奥林巴斯</td><td>扫描^ R分析软件（v.1.2.0.6） </td><td></td></tr></tbody></table>

studying proteolysis of cyclin b at the single cell level in whole cell populations

两个子细胞在细胞分裂过程中染色体之间平等分配的前提条件是保证遗传稳定性1。染色体分离过程中的不准确之处是恶性肿瘤的一个标志，并伴有渐进性疾病2-4。有丝分裂纺锤体组装检查点（SAC）是一个监督机制，阻碍直到每一个染色体的细胞在中期建立了稳定的双极连接到有丝分裂纺锤体1。 SAC发挥其功能的干扰与激活APC / C亚基CDC20阻止蛋白水解的securin和细胞周期蛋白B和染色体分离和有丝分裂退出。不当安装的染色体防止沉默的SAC的信号，导致继续抑制APC / C CDC20，直到问题得到解决，以避免染色体分离现象的原因，非整倍体和恶性生长。 在我的大多数研究APC / C-依赖的蛋白降解的影响及的不当染色体附件上利用了主轴中断使用微管解聚或稳定的药物来干扰微管与染色体附件。由于干扰微管动力学影响的运输和国产化的关键调节，这些程序承担的风险，导致人为影响5。 要研究如何SAC干扰APC / C-依赖的蛋白降解的细胞周期蛋白B的有丝分裂过程中泰然自若的细胞群，我们建立了组蛋白H2-GFP为基础的系统，该系统允许同时监测中期调整的有丝分裂染色体的细胞周期蛋白B 6和蛋白水解。 要描绘的蛋白水解的公司，我们产生了嵌合细胞周期蛋白B，报道分子的C-末端的SNAP部分6（ 图1）。 SNAP-部分是在一个自标记反应，能够形成共价键烷基鸟嘌呤-载体（SNAP基板）7,8（ 图1）。 SNAP底物分子都是现成的，进行广泛的不同的荧光染料。嵌合细胞周期蛋白B-SNAP分子成为膜可渗透SNAP衬底的生长介质7（ 图1）添加后标记。荧光强度的细胞周期蛋白B-SNAP标记反应后，在一个脉冲追踪反应类似的方式和荧光强度反映细胞周期蛋白B降解6（ 图1）的水平的下降。我们的系统有利于在大量的细胞（或多个细胞群）在平行的有丝分裂，APC / C-依赖的蛋白降解的监测。由此，该系统可以是一个有价值的工具，以确定代理/小分子，能够干扰蛋白水解活性，在中期到后期过渡。此外，作为在有丝分裂过程中合成的细胞周期蛋白B最近已被建议作为一个重要mechanis米，在小鼠和人类保持cyclin B的表达水平基本稳定9,10促进有丝分裂块，这个系统使我们能够分析细胞周期蛋白B蛋白水解的一个元素平衡的平衡6。

Watch this Scientific Journal Video about Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations at JoVE.com

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

中期向后期过渡被触发，通过后期促进复合物（APC / C）依赖的泛素化和随后的破坏细胞周期蛋白B.在这里，我们建立了一个系统，脉冲追踪标签，可以监控在整个细胞群的细胞周期蛋白B蛋白水解和有利于检测干扰的有丝分裂关卡。

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

studying-proteolysis-cyclin-b-at-single-cell-level-whole-cell

Research

JoVE Journal

Biology

10.4K 次观看.  University Medical Center Freiburg. 中期向后期过渡被触发，通过后期促进复合物（APC / C）依赖的泛素化和随后的破坏细胞周期蛋白B.在这里，我们建立了一个系统，脉冲追踪标签，可以监控在整个细胞群的细胞周期蛋白B蛋白水解和有利于检测干扰的有丝分裂关卡。

视频: Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

全细胞记录的光在视网膜切片诱发兴奋性突触电流

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

科研

生物学

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

人死后脑组织中的单细胞群获得高品质的RNA

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

的分散Presomitic中胚层细胞培养的斑马鱼分割时钟在单细胞成像代

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.