Name: determination of lipid raft partitioning of fluorescently-tagged probes in living cells by fluorescence correlation spectroscopy (fcs)
Uploaded: 2012-04-06T13:00:00
Description: Watch this Scientific Journal Video about Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS at JoVE.com

这项工作得到了从法新社德拉国民RECHERCHE（ChoAD“）的资助。我们也感谢基金会的财政支持ICM（研究所杜Cerveau ET DE LA Moelle）。

1。 FCS的安装校准<ol><li>启动共聚焦显微镜，激光，计算机，孵化温度和CO 2控制。 </li><li>确保SPAD值（单光子雪崩二极管）和荧光过滤器内的SPAD非常适合你的样品。检查的SPAD时间同步。小心才开始您的FCS的软件，一旦你的SPAD设置为收购准备。 </li><li>准备一个新鲜的霍乱毒素-Alexa488解决方案，在PBS稀释至浓度达到1微克/毫升（17.5海里）。 </li><li>优化解决方案的使用显微镜的内部检测的?...

<ol>
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这里介绍FCS的方法，使脂筏的荧光探针在活细胞中的利益分割的敏感和快速分析。 FCS结合共聚焦显微镜，单光子计数的灵敏度的定位精度。 FCS和标准的生化技术之间的主要区别是，FCS使脂筏的目标，而不是相对分区的分区DRMS隔离或合作修补的情况下是绝对测定。 FCS的数据采集每个样品约5分钟（30秒10的收购），因此是相当迅速，比一般的生化技术。这个速度让时间有可能?...

<html><table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>霍乱毒素B亚基的网站488 </td><td> Invitrogen公司</td><td>的C-34775 </td><td>兆瓦（五聚）= 57千克/摩尔</td></tr><tr><td>共聚焦显微镜</td><td>徕卡</td><td> SP5的</td><td></td></tr><tr><td>孵化器温度和CO 2控制</td><td>生活影像服务</td><td>立方盒</td><td></td></tr><tr><td> SPAD值（单光子雪崩二极管） </td><td> MPD公司（微型光子器件） </td><td> PDM系列（100微米的敏感区） </td><td></td></tr><tr><td>高通488纳米过滤器</td><td> semrock </td><td> 488纳米，阻断边缘BrightLine长通滤波器 部分＃ails.aspx？= FF01-488/LP-25“目标=”_blank“&gt; FF01-488/LP-25 </td><td></td></tr><tr><td> FCS检测单位</td><td> picoquant </td><td> picoharp 300模块</td><td></td></tr><tr><td>采集和自动相关软件</td><td> picoquant </td><td> SymPhoTime </td><td></td></tr><tr><td>拟合软件</td><td> OriginLab </td><td> OriginPro8 </td><td></td></tr></tbody></table>

determination of lipid raft partitioning of fluorescently-tagged probes in living cells by fluorescence correlation spectroscopy (fcs)

在过去15年中，细胞膜是不是均匀的，并依赖于微，以发挥其职能的概念已成为被广泛接受。脂筏中富含胆固醇和鞘脂膜微。他们发挥的作用，在细胞的生理过程，例如信号和贩运1,2，但也被认为是几种疾病，包括病毒或细菌感染和神经退行性疾病中的关键球员。 然而，它们的存在仍是一个争论的问题4,5。事实上，脂筏的大小已估计为20纳米左右6，远低于传统的显微镜（约200纳米）的分辨率极限，从而解除了他们的直接成像。到现在为止，用于评估蛋白质脂筏内的利益分割的主要技术洗涤剂耐膜（DRMS）抗体的分离与合作修补。尽管广泛使用贝科使用他们比较容易实现的，这些技术容易文物，从而批评7,8。因此，有必要克服这些文物，能够探测活细胞中的脂筏分区技术改进。 在这里，我们提出了一个敏感的荧光标记的蛋白质在活细胞的细胞膜脂质的脂筏分区分析方法。这种方法，被称为荧光相关光谱（FCS），依赖于在位于内部或外部的脂筏的荧光探针的扩散时间的差距。事实上，在人工膜和细胞培养证明，探测器将扩散速度比内致密的脂质筏9,10外。分钟的荧光波动来确定扩散时间，测量的时间在一个焦点量（约1 femtoliter）的功能，用共聚焦显微镜在位于细胞质膜（ 图1）。自相关曲线，然后从这些波动，并安装适当的数学扩散模型11。 FCS可以用来确定分割的各种探针，只要他们有荧光标记的脂质筏。荧光融合蛋白的表达或荧光配体的结合，可以实现荧光标记。此外，FCS可以使用不仅在人工膜和细胞系，而且在小学文化，描述最近12。它也可以用来追踪脂筏的动态分区后，除了药物或膜脂组成的变化12。

Watch this Scientific Journal Video about Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS at JoVE.com

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

描述一种技术来探测荧光蛋白在活细胞的细胞膜脂筏分区。它发生在位于内部或外部的脂筏蛋白的扩散时间的悬殊优势。收购可以进行动态控制条件或吸毒后。

脂筏的测定分区在活细胞中的荧光标记探针的荧光相关光谱（FCS）

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Research

JoVE Journal

Biology

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

Measuring the Kinetics of mRNA Transcription in Single Living Cells

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

科研

生物学

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

绿色荧光蛋白标记的质膜蛋白质的扩散系数的简易测量采用K空间图像相关光谱

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

采用Caco-2细胞研究脂质运输由肠

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in 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 ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

共聚焦显微镜通过图像相关谱揭示细胞表面受体聚集

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...