Name: assaying surface expression of chemosensory receptors in heterologous cells
Uploaded: 2011-02-23T17:30:00
Description: Watch this Scientific Journal Video about Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells at JoVE.com

我们感谢阅读这篇稿子的关键松波实验室的成员。

1。程序： 该过程包括三个部分，超过3天的总时间完成：转移细胞，转染和免疫细胞化学，每天进行一个步骤。转移和细胞的转染前两天，必须在层流室的无菌条件下进行。 第1天：转让HEK293T细胞表面表达检测。 <ol><li>传输介质（M10）：混合辅以10％（体积比）牛血清胎儿在无菌条件下的最低限度的基本介质。 </li><li>种植细胞所需的汇合在一个100mm的培养...

<ol>
	<li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65, 175-187 (1991).</li><li>Malnic, B., Hirono, J., Sato, T., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+receptor+codes+for+odors.">Combinatorial receptor codes for odors.</a> Cell. 96, 713-723 (1999).</li><li>Saito, H., Kubota, M., Roberts, R. W., Chi, Q., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTP+family+members+induce+functional+expression+of+mammalian+odorant+receptors.">RTP family members induce functional expression of mammalian odorant receptors.</a> Cell. 119, 679-691 (2004).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell-surface+expression+and+measuring+activation+of+mammalian+odorant+receptors+in+heterologous+cells.">Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.</a> Nat Protoc. 3, 1402-1413 (2008).</li></ol>

每一步都必须小心进行，以确保突出和独特的表面染色。整个染色过程中，必须在寒冷的开展（冰），和盖玻片奠定托盘必须被冷却之前使用，以确保细胞活路。此外，固定做表面染色过程结束。这是内部染色，其中固定先染色通透细胞膜的小学和中学抗体形成鲜明对比的。盖玻片暴露在空气中的时间必须最小化，以防止从干涸的细胞，因此盖玻片与抗体的解决方案的分层，转让盖玻片洗涤液，?...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Cover slip 22 X 22 mm (#1)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>72200-11</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>16000</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
<td>&nbsp;</td>
<tr>
<td>Minimal essential medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M4655</td>
<td>With L glutamate, Earle&#x2019;s salt and bicarbonate</td>
</tr>
<tr>
<td>Mowiol</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>475904</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>EMS</td>
<td>19208</td>
<td>&nbsp;</td>
<tr>
<td>Hanks Balanced Salt Solution (HBSS) 1X </td>
<td>&nbsp;</td>
<td>Gibco </td>
<td>14025</td>
<td>With calcium chloride and magnesium chloride</td>
</tr>
<tr>
<td>HEPES Buffer Solution</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffer Saline (PBS)</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-040-CV</td>
<td>Without calcium and magnesium</td>
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<tr>
<td>Poly D Lysine Hydrobromide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Azide</td>
<td>&nbsp;</td>
<td>EM Science</td>
<td>SX0299-1</td>
<td>&nbsp;</td>
<tr>
<td>Tissue culture dish 35 X 10 mm</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>353801</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>0.05%</td>
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assaying surface expression of chemosensory receptors in heterologous cells

是公认的嗅觉感的生动的气味世界。在小鼠的嗅觉是介导的由约1200克蛋白偶联受体（GPCRs）1，假设绑定挥发的气味分子，并通过与G蛋白耦合Gαolf的胞内信号转换成细胞外信号的剧目。绑定的气味受体被认为是按照组合规则，也就是说，一个气味可能几个受体绑定和一个受体可以绑定几个气味不同程度2。生化，信令和配体结合研究已方便进行最GPCRs的使用异种细胞。但是使用的气味受体的研究，异种细胞，排除了很长一段时间，因为他们在转没有出口到表面。 Saito等人已经证明单膜通过受体运输蛋白（RTP）的家庭伴侣显示在嗅感觉神经元和交通气味受体在异种细胞的表面，当联合一起转了3伴侣的行为表达增强。要利用异种细胞的受体进行生化检测，必须先确定，如果受体的细胞株显示了强大的表面表达。这可以检测高表达的受体与由活细胞染色，荧光标签或专门的外域外域标记的伴侣RTP1S。在这里，我们展示了一个协议，进行活细胞染色，可用于检测HEK293T细胞表面上的气味受体方便。此外，它也可能被用来检测其他化学感受受体或GPCRs的表面表达。

Watch this Scientific Journal Video about Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells at JoVE.com

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

在这里，我们展示了一个协议，进行活细胞染色，可用于检测HEK293T细胞表面上的气味受体方便。此外，它也可能被用来检测其他化学感受受体或GPCRs的表面表达。

测定在异种细胞表面的化学感应受体的表达

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

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Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

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Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

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The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

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Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...