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）
Summary
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。
Abstract
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。
Protocol
1。 FCS的安装校准启动共聚焦显微镜，激光，计算机，孵化温度和CO 2控制。 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。 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）。 优化解决方案的使用显微镜的内部检测的?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Cholera toxin subunit B-Alexa 488 | Invitrogen | C-34775 | MW (pentamer) = 57 kg/mol |
| Confocal microscope | Leica | SP5 | |
| Incubator for temperature and CO2 control | Life imaging services | The Cube and the Box | |
| SPAD (Single Photon Avalanche Diode) | MPD (Micro Photon Devices) | PDM serie (100 μm sensitive area) | |
| High pass 488 nm filter | Semrock | 488 nm blocking edge BrightLine long-pass filter Part # FF01-488/LP-25 |
|
| FCS detection unit | Picoquant | Picoharp 300 module | |
| Acquisition and auto-correlation software | Picoquant | SymPhoTime | |
| Fitting software | OriginLab | OriginPro8 |
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Tags
Lipid Raft PartitioningFluorescently-tagged ProbesLiving CellsFluorescence Correlation SpectroscopyCell MembranesMicrodomainsCholesterolSphingolipidsSignallingTraffickingDiseasesControversyLipid Raft SizeMicroscopy Resolution LimitDirect ImagingTechniquesDetergent Resistant MembranesCo-patching With AntibodiesArtefactsSensitive AnalysisPlasma Membrane
Citar este artigo
Marquer, C., Lévêque-Fort, S., Potier, M. Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS). J. Vis. Exp. (62), e3513, doi:10.3791/3513 (2012).