Name: live imaging of dense-core vesicles in primary cultured hippocampal neurons
Uploaded: 2009-05-29T00:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons at JoVE.com

我们感谢他们仔细阅读这篇稿子哈拉尔胡特尔和海伦娜德克尔。我们也感谢徕卡Microsystems的注册Sidhu，他的技术专长。这项研究是由美国国家科学与工程理事会，加拿大，奖＃327100-06，西蒙弗雷泽大学理学院的支持。

第1部分：用脂质体2000（Invitrogen）的神经元的转设备设置： 培养的大鼠或小鼠海马神经元是根据Kaech和银行家，2006 [4]。典型的转染的细胞密度为25万cells/6-cm菜。所需的试剂和设备，包括50 mM的kynurenic酸，定期台式管支架，台式冷却器（最好保持脂质体转染试剂冷）， 脂质体转染试剂，MEM，离心管，micropipetters和无菌镊子。 程序： <o...

<ol>
	<li>Kulic, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+microtubule+movement+in+bidirectional+organelle+transport.">The role of microtubule movement in bidirectional organelle transport.</a> Proc Natl Acad Sci U S A. 105 (29), 10011-10016 (2008).</li><li>Jacobson, C., Schnapp, B., Banker, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+change+in+the+selective+translocation+of+the+Kinesin-1+motor+domain+marks+the+initial+specification+of+the+axon.">A change in the selective translocation of the Kinesin-1 motor domain marks the initial specification of the axon.</a> Neuron. 49 (6), 797-804 (2006).</li><li>Barkus, R. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+Axonal+Kinesin-3+Motor+for+Fast+Anterograde+Vesicle+Transport+that+Facilitates+Retrograde+Transport+of+Neuropeptides.">Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides.</a> Mol Biol Cell. 19 (1), 274-283 (2008).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1 (5), 2406-2415 (2006).</li></ol>

活细胞成像是一个具有挑战性，但强大的技术，直接观察培养的神经元细胞器运输。困难可能会出现上游与神经元的健康欠佳，由于文化并发症程序。因此，细胞的健康（例子见文献4）应评估转染前后，而在生长介质中使用组织培养，光镜下观察盖玻片。转移过程中的神经元含有盖玻片成像室，可紧张的细胞，因此重要的是要小心操纵的盖玻片。它可以是共同的，健康的细胞没有运输过程中的延...

A correction was made to <a href="http://www.jove.com/details.php?ID=1144">Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons</a>. There was an error in the author's name. The author name was corrected to include a middle initial as follows:

David M. Kwinter, Michael A. Silverman

instead of:

David Kwinter, Michael Silverman.

A correction to author names has been made for article Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.

Erratum: Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons.

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<td>MEM-Eagle with Earle salts and L-glutamine</td>
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<td>Mediatech</td>
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<td>&nbsp;</td>
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<td>Lipofectamine 2000</td>
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<td>10X Hanks with Ca2+ and Mg2+</td>
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<td>14185-052</td>
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<td>1M HEPES</td>
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live imaging of dense-core vesicles in primary cultured hippocampal neurons

动态细胞过程的观测和特征，可以产生对细胞活性，不能从静态图像获得的重要信息。重要的荧光探针，尤其是绿色荧光蛋白（GFP）已经彻底改变了细胞生物学能力标签特定的细胞内的车厢和细胞结构产生。例如，允许实时成像的GFP（和其光谱变种）嵌合体的细胞骨架，细胞器运输，并在众多生物和细胞类型[1-3]膜动态动态分析。虽然实时成像已成为普遍的，这种做法仍然引起了许多技术挑战，特别是在原代培养的神经元，。挑战之一是GFP标记的蛋白质在有丝分裂后神经元的表达，另一种是能够捕捉到的荧光图像，同时最大限度地减少光毒性，漂白，并保持细胞的健康一般。在这里，我们提供了一个协议，它描述了一个脂质体转染法，收益率相对较低的转染率（0.5％），但是是完全极化神经元成像的理想选择。低转染率是至关重要的，使单个轴突和树突细胞体内的方向的特点，以确认的方向性，即运输，顺行诉逆行。我们的方法来成像GFP的表达神经元依赖于一个标准的宽视场荧光显微镜与CCD摄像头，图像采集软件，和热成像室配备。我们有成像的细胞器或结构的种类繁多，例如，没有任何特殊的光学或激发需求，比荧光灯光源的致密核心囊泡，线粒体，生长锥，与肌动蛋白。此外，不同的光谱，荧光标记的蛋白质，例如，绿色荧光蛋白和蛋白DsRed的标签，可附近同时可视化表征的合作运输或其他协调的细胞活动。成像方法，这里描述的是各种成像应用灵活，可以由实验室通过相对较少的成本提供了一个显微镜。

Watch this Scientific Journal Video about Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons at JoVE.com

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

活细胞成像研究细胞器官贩卖的动态时，特别实用。在这里，我们描述了一个致密核心囊泡在培养的神经元，采用宽视场荧光显微镜的实时成像的协议。此协议是灵活的和可适应图像的其它细胞器如线粒体，内体，和过氧化物酶。

在原代培养海马神经元的密集核心囊泡的实时成像

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

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从P0新生大鼠海马神经元的原代培养

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

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Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

4D Imaging of Protein Aggregation in Live Cells

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

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