Name: imaging phluorin-tagged receptor insertion to the plasma membrane in primary cultured mouse neurons
Uploaded: 2012-11-20T13:00:00
Description: Watch this Scientific Journal Video about Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons at JoVE.com

这项工作是从杰克逊实验室的启动资金支持。

1。准备鼠神经胶质细胞培养的神经元培养空调<ol><li>涂有胶原蛋白溶液（1:3稀释Purecol DDH 2 O中）的T75烧瓶。烧瓶一身正气，干通宵的组织文化引擎盖。 </li><li>夹层缓冲液（人工脑脊液：119 mM氯化钠，5 mM KCl中，1毫摩尔MgCl 2和30mM葡萄糖，25mM的HEPES，pH值7.4，无钙）和神经胶质细胞介质（DMEM培养液与10％胎牛血清，10单位/ ml青霉素，10 μg/ ml链霉素，和Glutamax）的制备和贮存在4℃下?...

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Neurosci. 27, 13706-13718 (2007).</li><li>Makino, H., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+incorporation+into+synapses+during+LTP:+the+role+of+lateral+movement+and+exocytosis.">AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis.</a> Neuron. 64, 381-390 (2009).</li><li>Makino, H., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+versus+global+synaptic+plasticity+on+dendrites+controlled+by+experience.">Compartmentalized versus global synaptic plasticity on dendrites controlled by experience.</a> Neuron. 72, 1001-1011 (2011).</li><li>Wang, Z., Edwards, J. G., Riley, N., Provance, D. W., Karcher, R., Li, X. D., Davison, I. G., Ikebe, M., Mercer, J. A., Kauer, J. A., Ehlers, M. 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不知什么原因，小鼠神经元总是很难培养的大鼠神经元。在我们的经验中，混合培养的神经元和神经胶质细胞的原代培养的小鼠神经元。然而，这样的混合培养TIRF成像实验是不适合的，在这种类型的培养的神经元和其流程往往顶部的神经胶质细胞上生长的情况，神经元的胞体和树突状进程鞭长莫及TIRF显微镜。因此，较低的密度盖玻片上少数神经胶质细胞的神经元培养TIRF成像的理想选择。我们首?...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt;试剂名称</strong</td&gt;<td<strong&gt;公司</strong</td&gt;<td<strong&gt;产品目录号</strong</td&gt;<td<strong&gt;评论</strong</td&gt;</tr&gt;<tr&gt;<td&gt;纯化的牛胶原溶液（Purecol）</td&gt;<td&gt;“高级”BIOMATRIX</td&gt;<td&gt; 5005-B</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Hank氏平衡盐溶液（HBSS）</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 14185-045</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td青霉素，链霉素（笔链球菌）</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 15140-122</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;丙酮酸钠</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 11360-070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM高糖</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 10313-021</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;牛胎儿血清（FBS）的</td&gt;<td</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; GlutaMAX</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 35050-061</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;木瓜蛋白酶</td&gt;<td沃辛顿生化公司</td&gt;<td&gt; LS003126</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;从牛胰脱氧核糖核酸酶Ⅰ（DNA酶I）</td&gt;<td&gt; SIGMA</td&gt;<td&gt; DN25-10MG</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Dulbecco氏磷酸盐缓冲盐水（DPBS）</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 14190-144</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 0.05％胰蛋白酶</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 25300-054</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td聚-L-赖氨酸氢溴酸盐</td&gt;<td&gt; SIGMA</td&gt;<td&gt; P2636-1G</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;硼酸</td&gt;<td&gt;费舍尔的科学</td&gt;<td&gt; BP 168-500</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Neurobasal培养基</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 21103-049</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; B-27的无血清补充</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 17504-044</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;热不活动的马血清</td&gt;<td&gt; GIBCO</td&gt;<td&gt; 26050-070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;脂质体2000</td&gt;<td&gt; Invitrogen公司</td&gt;<td&gt; 11668 019</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; HEPES</td&gt;<td&gt;费舍尔的科学</td&gt;<td&gt; BP310-500</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt;企业文化插入</td&gt;<td&gt;密理博</td&gt;<td&gt; PICM03050</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

imaging phluorin-tagged receptor insertion to the plasma membrane in primary cultured mouse neurons

质膜（PM）和细胞内受体之间贩运的机制更好地理解需要具有出色的空间分辨率和时间分辨率的实验方法。此外，这种方法还必须有分辨能力定位于PM在细胞内的受体。最重要的是，检测的受体，在一个单一的囊泡需要优异的检测灵敏度，因为每个囊泡进行只有一小数量的受体。检查受体活动的标准方法，包括表面生物素的生化检测，缺乏必要的空间和时间的决议;和荧光显微镜检查的immunolabeled表面的受体，这需要细胞化学固定，因此缺乏足够的时间分辨率1-6。为了克服这些限制，我们和其他人开发的，采用了一种新STRAtegy，使可视化动态插入的受体出色的空间分辨率和时间分辨率7-17 PM。该方法包括pH敏感的GFP，superecliptic的pHluorin 18的N-末端胞外结构域的受体的标记。 superecliptic pHluorin荧光在中性pH值和非荧光在酸性pH值（pH值&lt;6.0）的独特属性。因此，标记受体的非荧光内酸性细胞内转运囊泡管腔或内体舱室时，它们变得容易地可视化，只有当暴露于细胞外的中性pH值环境上的外表面的PM。因此，我们的战略使我们能够区分PM表面受体，在细胞内囊泡。为了达到足够的空间和时间分辨率，以及学习所需的动态贩卖受体的敏感性，我们采用全内反映离子荧光显微镜（TIRFM），这使我们能够实现光学成像（〜170 nm）的最佳的空间分辨率，时间分辨率的视频率显微镜（30帧/秒），且灵敏度来检测单个GFP的荧光分子。成像pHluorin标记的受体TIRFM下，我们能够直接可视个人到PM培养的神经元受体插入事件。这种成像方法可能被应用于任何膜蛋白的胞外结构域，可以标记superecliptic pHluorin，并且将允许的密钥的详细机制不同的膜蛋白（受体，离子通道，运输等）插入到夹层PM。

Watch this Scientific Journal Video about Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons at JoVE.com

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

通过标记与superecliptic pHluorin膜受体的胞外域的，并通过成像这些融合在培养的小鼠神经元受体，我们可以直接可视化个别水泡插入事件到质膜的受体。这种技术将有助于阐明受体插入到质膜的分子机制。

成像pHluorin标记插入到质膜受体原代培养的小鼠神经元

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Research

JoVE Journal

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

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After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Primary Culture of Mouse Dopaminergic Neurons

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...