基于细胞中钙含量TRP通道功能的高通量筛选，使用FlexStation 3
Summary
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。
Abstract
“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。
Protocol
1。在96孔板细胞的准备 HEK293细胞与人类TRPA1基因瞬时trasfected 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％。 大衣96孔板吹打，每孔50μL聚- L -?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| FlexStation 3 | Molecular Devices | FLEX3 | |
| 96-Well, FlexStation Pipet Tips | Molecular Devices | 9000-0912 | |
| 96-Well Plates | Greiner Bio-one | 655090 | |
| 96-Well Clear Flat Bottom plates | Costar | 3595 | |
| DMEM | Invitrogen | 12430-104 | |
| Opti-MEM | Invitrogen | 31985-070 | |
| Lipofectamine 2000 | Invitrogen | 11668-019 | |
| Fluo-4 AM | Invitrogen | F14202 | |
| PluronicF-127 | Invitrogen | P3000MP | |
| Poly-L-ornithine | Sigma | P36551 | |
| probenecid | Sigma | P8761 | |
| Bovine serum albumin | Sigma | A9418 |
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Tags
Cell-based Calcium AssayMedium To High Throughput ScreeningTRP Channel FunctionsFlexStation 3Multi-mode Microplate ReaderFluorescence MeasurementMulti-well PlatesAcademic SettingsIntegrated Fluid Transfer ModuleMulti-channel PipetterFluorescence ChangesCa2+-permeable Ion ChannelsG-protein Coupled ReceptorsIntracellular Free Ca2+ LevelsTransient Receptor Potential (TRP) ChannelsCalcium InfluxTRPA1 ChannelMolecular SensorNoxious StimuliHEK293 CellsCa2+-sensitive Fluorescent DyeFluo-4Real-time Fluorescence ChangesTRPA1 AgonistTRPA1 Antagonist
Cite This Article
Luo, J., Zhu, Y., Zhu, M. X., Hu, H. Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3. J. Vis. Exp. (54), e3149, doi:10.3791/3149 (2011).