Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures.
Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures.
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原代神经元培养物中膜神经递质转运蛋白的抗体偶联单量子点追踪。
DOI：
10.1007/978-1-4939-6840-4_11
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Rosenthal,SandraJ
中科院分区：
文献类型：
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作者：
Bailey,DanielleM;Kovtun,Oleg;Rosenthal,SandraJ
单粒子追踪（SPT）实验为科学界提供了关于个体受体、转运蛋白、激酶、脂质和分子马达动态调节的宝贵单分子信息。SPT是系综平均方法的替代方法，其中可能丢失异构运动模式。量子点具有光稳定性好、亮度高、发射光谱窄、尺寸依赖等优点，是SPT实验的理想探针。在典型的基于QD的SPT实验中，QD与感兴趣的目标结合，并通过荧光视频显微镜成像数秒至数分钟。然后将各个帧中的单个QD点链接以形成轨迹，分析这些轨迹以确定它们的均方位移、扩散系数、约束指数和瞬时速度。本章描述了一个可推广的协议，用于单粒子跟踪细胞膜上的膜神经递质转运蛋白与未修饰的细胞外抗体探针和二级抗体偶联量子点或生物素化的细胞外抗体探针和链霉亲和素偶联量子点在原代神经元培养。讨论了神经元细胞培养、生物素化方案和量子点标记程序以及基本数据分析。
Single particle tracking (SPT) experiments have provided the scientific community with invaluable single-molecule information about the dynamic regulation of individual receptors, transporters, kinases, lipids, and molecular motors. SPT is an alternative to ensemble averaging approaches, where heterogeneous modes of motion might be lost. Quantum dots (QDs) are excellent probes for SPT experiments due to their photostability, high brightness, and size-dependent, narrow emission spectra. In a typical QD-based SPT experiment, QDs are bound to the target of interest and imaged for seconds to minutes via fluorescence video microscopy. Single QD spots in individual frames are then linked to form trajectories that are analyzed to determine their mean square displacement, diffusion coefficient, confinement index, and instantaneous velocity. This chapter describes a generalizable protocol for the single particle tracking of membrane neurotransmitter transporters on cell membranes with either unmodified extracellular antibody probes and secondary antibody-conjugated quantum dots or biotinylated extracellular antibody probes and streptavidin-conjugated quantum dots in primary neuronal cultures. The neuronal cell culture, the biotinylation protocol and the quantum dot labeling procedures, as well as basic data analysis are discussed.