Name: cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3
Uploaded: 2011-08-17T12:00:00
Description: Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

我们感谢博士。约翰汉考克，雷烧烤，安德鲁莫里斯和奥尔加Chumakova的支持。 FlexStation 3系统UTHSC Cytodynamic成像设施的一部分。我们也感谢博士。 Ardem Patapoutian和迈克尔Bandell来自斯克里普斯研究所和诺华研究基金会（GNF）的基因组研究所提供的HEK293 - hTRPA1稳定的细胞株。支持这项研究是由NIH（GM081658 MXZ）和德州医学中心消化内科中心（HH）和使命连接/ TIRR基金会（农户）的赠款部分。

1。在96孔板细胞的准备 HEK293细胞与人类TRPA1基因瞬时trasfected <ol><li> HEK293细胞成长为一到两天贝科的最小必要中等含有4.5 mg / ml的葡萄糖（Thermo Scientific的，SH3002201）（DMEM），辅以10％热灭活胎牛血清（Invitrogen公司，16000-044），50个单位/ ml青霉素和50μg/ mL链霉素（Invitrogen公司，15140-163）。在一天的转染，细胞密度应达到70-90％。 </li><li>大衣96孔板吹打，每孔50μL聚- L -?...

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	<li>Hu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+TRPA1+channels+by+fenamate+nonsteroidal+anti-inflammatory+drugs.">Activation of TRPA1 channels by fenamate nonsteroidal anti-inflammatory drugs.</a> Pflugers Arch. 459, 579-592 (2010).</li><li>Hu, H., Bandell, M., Petrus, M. J., Zhu, M. X., Patapoutian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+activates+damage-sensing+TRPA1+ion+channels.">Zinc activates damage-sensing TRPA1 ion channels.</a> Nat Chem Biol. 5, 183-190 (2009).</li><li>Perney, T. M., Hirning, L. D., Leeman, S. E., Miller, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+calcium+channels+mediate+neurotransmitter+release+from+peripheral+neurons.">Multiple calcium channels mediate neurotransmitter release from peripheral neurons.</a> Proc Natl Acad Sci. 83, 6656-6659 (1986).</li><li>Mason, S. A., MacLeod, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+action+potential+duration+and+calcium+regulation+in+males+and+females.">Cardiac action potential duration and calcium regulation in males and females.</a> Biochem Biophys Res Commun. 388, 565-570 (2009).</li><li>Raat, N. J., Wetzels, G. E., DeMey, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-contraction+relationship+in+rat+mesenteric+arterial+smooth+muscle.+Effects+of+exogenous+and+neurogenic+noradrenaline.">Calcium-contraction relationship in rat mesenteric arterial smooth muscle. Effects of exogenous and neurogenic noradrenaline.</a> Pflugers Arch. 436, 262-269 (1998).</li><li>Alagarsamy, S., Johnson, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+calcium+channel+involvement+in+NMDA-induced+activation+of+NOS.">Voltage-dependent calcium channel involvement in NMDA-induced activation of NOS.</a> Neuroreport. 6, 2250-2254 (1995).</li><li>Su, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM7+regulates+cell+adhesion+by+controlling+the+calcium-dependent+protease+calpain.">TRPM7 regulates cell adhesion by controlling the calcium-dependent protease calpain.</a> J Biol Chem. 281, 11260-11270 (2006).</li><li>Dubinsky, J. M., Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+concentrations+during+"chemical+hypoxia"+and+excitotoxic+neuronal+injury.">Intracellular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury.</a> J Neurosci. 11, 2545-2551 (1991).</li><li>George, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+channel+activation+augments+NMDA+receptor+function+and+promotes+neurite+outgrowth+in+immature+cerebrocortical+neurons.">Sodium channel activation augments NMDA receptor function and promotes neurite outgrowth in immature cerebrocortical neurons.</a> J Neurosci. 29, 3288-3301 (2009).</li><li>Price, K. L., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FlexStation+examination+of+5-HT3+receptor+function+using+Ca2++-+and+membrane+potential-sensitive+dyes:+advantages+and+potential+problems.">FlexStation examination of 5-HT3 receptor function using Ca2+ - and membrane potential-sensitive dyes: advantages and potential problems.</a> J Neurosci Methods. 149, 172-177 (2005).</li><li>Laude, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+functional+assays+of+recombinant+IP3+receptors.">Rapid functional assays of recombinant IP3 receptors.</a> Cell Calcium. 38, 45-51 (2005).</li><li>Marincsak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analgesic+drug,+tramadol,+acts+as+an+agonist+of+the+transient+receptor+potential+vanilloid-1.">The analgesic drug, tramadol, acts as an agonist of the transient receptor potential vanilloid-1.</a> Anesth Analg. 106, 1890-1896 (2008).</li><li>Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+high+throughput+screening+assays+against+three+subtypes+of+Ca(v)3+T-type+channels+using+molecular+and+pharmacologic+approaches.+Assay+Drug+Dev+Technol.">Validation of high throughput screening assays against three subtypes of Ca(v)3 T-type channels using molecular and pharmacologic approaches. Assay Drug Dev Technol.</a> 5, 191-203 (2007).</li><li>Christensen, B. N., Kochukov, M., McNearney, T. A., Taglialatela, G., Westlund, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-sensing+G+protein-coupled+receptor+mobilizes+calcium+in+human+synovial+cells.">Proton-sensing G protein-coupled receptor mobilizes calcium in human synovial cells.</a> Am J Physiol Cell Physiol. 289, C601-C608 (2005).</li><li>Boulay, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+a+novel+mammalian+homolog+of+Drosophila+transient+receptor+potential+(Trp)+involved+in+calcium+entry+secondary+to+activation+of+receptors+coupled+by+the+Gq+class+of+G+protein.">Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein.</a> J Biol Chem. 272, 29672-29680 (1997).</li><li>Nanda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+screening+of+adrenergic+receptors+by+measuring+intracellular+calcium+using+the+FlexStation+scanning+fluorimeter.">Functional screening of adrenergic receptors by measuring intracellular calcium using the FlexStation scanning fluorimeter.</a> Biotechnol J. 4, 417-422 (2009).</li></ol>

细胞内的Ca 2 +在许多生理和病理条件下，如神经递质的释放（3），心肌动作电位（4），肌肉细胞收缩（5），细胞信号转导（6-7）和兴奋性细胞死亡（，起着重要作用8）。细胞内Ca 2 +浓度的测量有助于澄清功能的变化质膜或细胞器中的Ca 2 +渗透的渠道的贡献 的Ca 2 +以上的流程（9-13） 。该检测也可以用于调查“G -蛋白偶联受体的功能，其中许多激活后...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> FlexStation 3 </td><td>分子器件</td><td> FLEX3 </td><td></td></tr><tr><td> 96孔，FlexStation吸取技巧</td><td>分子器件</td><td> 9000-0912 </td><td></td></tr><tr><td> 96孔板</td><td>格雷纳生物一</td><td> 655090 </td><td></td></tr><tr><td> 96，那么清除平底板</td><td>配角</td><td> 3595 </td><td></td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 12430-104 </td><td></td></tr><tr><td> OPTI - MEM </td><td> Invitrogen公司</td><td> 31985-070 </td><td></td></tr><tr><td>脂质体2000 </td><td> Invitrogen公司</td><td> 11668-019 </td><td></td></tr><tr><td>荧光凌晨4点</td><td> Invitrogen公司</td><td> F14202 </td><td></td></tr><tr><td> PluronicF - 127 </td><td> Invitrogen公司</td><td> P3000MP </td><td></td></tr><tr><td>聚- L -鸟氨酸</td><td>西格玛</td><td> P36551 </td><td></td></tr><tr><td>丙磺舒</td><td>西格玛</td><td> P8761 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A9418 </td><td></td></tr></tbody></table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

“分子器件FlexStation 3是一个台式多模酶标仪能够自动在多孔板荧光测量。它是理想的学术环境中高通量屏幕。它集成了一个流体输送模块，配备了多渠道pipetter机读取一列，在一个时间来监视各种荧光试剂的荧光变化。例如，FlexStation 3已被用于研究的Ca 2 +渗透离子通道和G蛋白偶联受体胞内游离Ca 2 +浓度的变化，通过测量的功能。瞬时受体电位（TRP）通道是一个大家族的非选择性阳离子通道，在许多生理和病理的功能发挥的重要作用。 TRP通道是钙离子渗透性和诱导激活后钙离子内流。在这段视频中，我们展示FlexStation 3的应用药理学研究TRPA1通道，为许多有害刺激的分子传感器。 HEK293细胞中瞬时或稳定表达人类TRPA1通道，生长在96孔板，装入一个的Ca 2 +敏感的荧光染料，荧光4，并在这些细胞中实时荧光变化的测量之前和期间的应用一个TRPA1受体激动剂，使用弹性模式FlexStation 3。一个公认的TRPA1受体拮抗剂的作用进行了研究。 SoftMax Pro软件的数据传送构建TRPA1激活剂和抑制剂的浓度 - 反应关系。

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

此视频提供了药理学研究，人类TRPA1使用FlexStation 3的渠道的详细协议。该协议包括细胞的准备，染料装载和酶标仪，FlexStation 3操作细节。

基于细胞中钙含量TRP通道功能的高通量筛选，使用FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

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