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Calcium Mobilization Assay to Measure the Activity of Gq-coupled Receptors
采用钙流检测法测定Gq偶联受体的活性   

编审
Cheng Zhang Cheng Zhang
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Abstract

Calcium mobilization assay is a cell-based second messenger assay to measure the calcium flux associated with Gq-protein coupled receptor activation or inhibition. The method utilizes a calcium sensitive fluorescent dye that is taken up into the cytoplasm of most cells. In some cell lines in which organic-anion transporters are particularly active (e.g. CHO, HeLa), addition of probenecid, an inhibitor of anion transport, is required for retention of this dye in the cells. The dye binds the calcium released from intracellular store and its fluorescence intensity increases. The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of the receptor of interest. This protocol can be applied to most mammalian cell lines expressing both endogenous and transiently/stably transfected receptors. The method is sensitive enough to be used for low-expressing systems or high throughput screening of target of interest.
Note: The method does not differentiate the Ca2+ mobilization induced by Gqα from the Ca2+ mobilization induced by Gβγ.

Keywords: GPCR(G蛋白偶联受体), Calcium(钙), Gq protein(Gq蛋白), FLIPR(FLIPR), AT1R(AT1R)

Materials and Reagents

  1. Cells of choice: HEK293 cells stably expressing Angiotensin II Type 1 Receptor (AT1R)
  2. Ligands of choice: Sar1-Angiotensin II (Bachem, catalog number: H1740 )
  3. Poly-L-lysine (Sigma-Aldrich, catalog number: P4707 )
  4. 1x phosphate-buffered saline (PBS) 
  5. FLIPR Calcium 5 assay kit (Molecular Devices, catalog number: R8185 )
  6. 96-well, FlexStation pipet tips, black (Molecular Devices, catalog number: 9000-0911 )
  7. Probenecid (Life Technologies, Invitrogen™, catalog number: P36400 )
  8. Cell growth media (see Recipes)

Equipment

  1. Incubator (5% CO2, 37 °C)
  2. FlexStation® 3 Multi-Mode Microplate Reader (Molecular Devices)
  3. Assay plate: 96-well microplate, tissue culture treated, black/clear, with lid (Greiner bio one, catalog number: 655090 )
  4. Ligand plate: Clear 96-well microtest plate, tissue culture treated, U-bottom (BD Biosciences, Falcon®, catalog number: 353077 )

Software

  1. SoftMax® Pro Microplate Data Acquisition & Analysis Software (supplied with the FlexStation® 3 Multi-Mode Microplate Reader)

Procedure


I.   Preparing the assay plate:

  1. Pre-coat the assay plate with 0.01% poly-L-lysine (50 μl/well) for 30 min at 37 °C (or 2 h at room temperature).
  2. Remove and wash excess poly-L-lysine with PBS.
  3. Prepare uniform suspension of cells of choice expressing the receptor and seed 50,000 cells per well in 50 μl medium.
    Notes:
    a. The cell number needs to be optimized for a particular cell line so that a 90% to 100% confluent cell monolayer is formed on the day of the assay.
    b. If the cell line is transiently transfected with the receptor of interest, it is useful to measure the expression level of the receptor on the plasma membrane.
  4. Incubate the cells overnight at 37 °C, 5% CO2.
  5. Aspirate the medium and add 100 μl serum-free medium into each well. Incubate the cells for 2 h at 37 °C in the CO2 incubator.
  6. Prepare the FLIPR loading dye by mixing 10 ml of component A with component B (see manufacturer's instructions for more details) and mix well.
    Note: If your cells require probenecid, add probenecid into the loading dye at a final working concentration of 2.5 mM. Probenecid by Invitrogen is water-soluble and dissolves quickly in assay buffer. Probenecid should be added freshly on the day of the experiment.
  7. Remove the assay plate from the CO2 incubator. Do not aspirate serum free medium. Add an equal volume (100 μl) of FLIPR loading dye into each well and incubate for 30 min at 37 °C, 5% CO2 followed by a 30 min incubation at room temperature if the assay will be performed at room temperature. The plate can be incubated at 37 °C, 5% CO2 for an hour if the assay will be performed at 37 °C.
    Note: The incubation time and temperature needs to be optimized for a particular cell line.
  8. After incubation transfer the assay plate to the FlexStation® 3 Multi-Mode Microplate Reader.
    Note: The temperature should be set to optimum assay condition.

 II.  Preparing the ligand plate:

  1. Prepare ligands (activator/inhibitor) at 5x concentration in 1x PBS in a final volume of 200 μl in ligand plate.
    Notes:
    a. You can test either one or several different concentrations of ligand of interest.
    b. You need to have at least duplicates of each ligand concentration to do a statistical analysis of the data.
    c. Plan your experiment carefully so that the ligand plate should be loaded by the time the cells are ready to be assayed.

III. Setting up the instrument (FlexStation® 3 Multi-Mode Microplate Reader):

  1. Place the assay plate, the ligand plate and the pipette tips as directed by the manufacturer's instructions.
  2. Open the SoftMax® Pro Microplate Data Acquisition & Analysis Software.
  3. Click on settings and select the FLEX mode. Recommended experimental setup parameters are given below:
    a. Read mode: Fluorescence
    b. Wavelengths: Ex: 485; Em: 525; Cut off: Auto (515)
    c. Sensitivity: High (Choose medium or low sensitivity if you expect very strong signals, especially with cell lines stably overexpressing the receptor.)
    d. Timing: 120 s reading with an interval of 2 sec (or choose accordingly)
    e. Assay plate type: 96 well Greiner blk/clr btm
    6) Wells to read: entire plate (or choose accordingly)
    f. Compound transfer:
    • Initial volume: 200 μl
    • Transfers: 1 (or choose accordingly for multiple transfers)
    • Pipette height: 220 μl (adjusted slightly more than initial volume)
    • Pipette volume: 50 μl (to make the final volume of the ligand in the assay plate as 1x)
    • Rate: 1
    • Time point: 18 sec (or choose accordingly)
    g. Compound source: Costar 96 well Ubtm clr. 3 ml
    h. Autocalibrate: on
    i. Autoread: off
  4. Read

Representative data



Figure 1. HEK293-AT1R cells stimulated with (well H1) and without (well A1) 1 μM Sar1-Angiotensin II at 18th second. The decrease in the signal with time is most likely resulted from receptor internalization.

Recipes

  1. Cell growth media (for HEK293 cells stably expressing Angiotensin II Type 1 Receptor (AT1R)) Dulbecco's modified eagle medium (DMEM) supplied with 10% fetal bovine serum (FBS). 

Acknowledgments

This work was supported by the National Institutes of Health grants HL47570 and HL115964 (Sadashiva Karnik, Ph. D.) and National Research Service Award HL007914 (Hamiyet Unal, Ph. D.).

References

  1. Unal, H., Jagannathan, R., Bhatnagar, A., Tirupula, K., Desnoyer, R. and Karnik, S. S. (2013). Long range effect of mutations on specific conformational changes in the extracellular loop 2 of angiotensin II type 1 receptor. J Biol Chem 288(1): 540-551.

简介

钙动员测定是基于细胞的第二信使测定法,用于测量与Gq蛋白偶联受体激活或抑制相关的钙通量。该方法使用钙敏感荧光染料,其被吸收到大多数细胞的细胞质中。在其中有机 - 阴离子转运蛋白特别活性的一些细胞系(例如CHO,HeLa)中,需要加入丙烯酸,阴离子转运抑制剂,以将该染料保留在细胞中。染料结合从细胞内储存释放的钙,其荧光强度增加。荧光强度的变化与响应感兴趣受体的配体激活释放到细胞质中的细胞内钙的量直接相关。该方案可以应用于表达内源性和瞬时/稳定转染受体的大多数哺乳动物细胞系。该方法足够敏感,可用于低表达系统或感兴趣的靶标的高通量筛选。
注意:该方法不区分Gqα诱导的Ca2 +动员与Gβγ诱导的Ca2 +动员。

关键字:G蛋白偶联受体, 钙, Gq蛋白, FLIPR, AT1R

材料和试剂

  1. 选择细胞:稳定表达血管紧张素II 1型受体(AT1R)的HEK293细胞
  2. 选择的配体:Sar - 血管紧张素II(Bachem,目录号:H1740)
  3. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P4707)
  4. 1×磷酸盐缓冲盐水(PBS)
  5. FLIPR Calcium 5测定试剂盒(Molecular Devices,目录号:R8185)
  6. 96孔,FlexStation吸移管尖端,黑色(Molecular Devices,目录号:9000-0911)
  7. Probenecid(Life Technologies,Invitrogen TM,目录号:P36400)
  8. 细胞生长培养基(参见配方)

设备

  1. 培养箱(5%CO 2,37℃)
  2. FlexStation ® 3多功能微孔板读数器(Molecular Devices)
  3. 测定板:96孔微孔板,组织培养处理,黑色/透明,带盖(Greiner bio one,目录号:655090)
  4. 配体板:澄清的96-孔微测试板,组织培养处理的U-底(BD Biosciences,Falcon ,目录号:353077)

软件

  1. SoftMax Pro微孔板数据采集& 分析软件(随FlexStation ® 3多模式酶标仪一起提供)

程序


I.   准备测定板:

  1. 用0.01%聚-L-赖氨酸(50μl/孔)在37℃预孵育测定板30分钟(或在室温下2小时)。
  2. 用PBS清除和洗涤多余的聚-L-赖氨酸。
  3. 准备均匀悬浮细胞的选择表示受体和种子50,000细胞每孔在50μl培养基。
    注意:
    a。 细胞数需要针对特定细胞系进行优化,以便在测定当天形成90%至100%汇合的细胞单层。
    b。 如果细胞系用感兴趣的受体瞬时转染,测量质膜上受体的表达水平是有用的。
  4. 在37℃,5%CO 2下孵育细胞过夜
  5. 吸出培养基,并添加100μl无血清培养基到每个孔。在CO 2培养箱中在37℃下孵育细胞2小时
  6. 通过将10ml的组分A与组分B(参见制造商的说明书)混合,制备FLIPR加载染料。
    注意:如果你的细胞需要丙磺舒,加入丙磺舒到加载染料中,最终工作浓度为2.5mM。 Probenecid由Invitrogen是水溶性的,并且在测定缓冲液中快速溶解。在实验当天,应该新鲜加入丙磺舒。
  7. 从CO 2培养箱中取出测定板。不要吸入无血清培养基。向每个孔中加入等体积(100μl)的FLIPR负载染料,并在37℃,5%CO 2下孵育30分钟,然后在室温下温育30分钟,如果该测定将是在室温下进行。如果分析将在37℃下进行,则可以将平板在37℃,5%CO 2孵育1小时。
    注意:孵化时间和温度需要针对特定细胞系进行优化。
  8. 孵育后将测定板转移到FlexStation 3多模式微板读数器 注意:温度应设置为最佳测定条件。

  II。 准备配体板:

  1. 准备配体(激活剂/抑制剂)在1x PBS中的5倍浓度,在配体板中的最终体积为200μl。
    注意:
    a。 您可以测试一种或几种不同浓度的感兴趣的配体。
    b。 您需要至少具有每个配体浓度的重复,以对数据进行统计分析。
    c。 请仔细计划您的实验,以便在细胞准备测定时加载配体板。

III。 设置仪器(FlexStation ® 3多模式酶标仪):

  1. 按照制造商的说明指导放置测定板,配体板和移液管吸头
  2. 打开SoftMax ® Pro Microplate Data Acquisition& 分析软件。
  3. 单击设置并选择FLEX模式。 推荐的实验设置参数如下:
    一个。 读取模式:荧光
    b。 波长:Ex:485; Em:525; 剪切:自动(515)
    C。 灵敏度:高(如果您期望信号非常强,请选择中等或低灵敏度,特别是细胞系稳定过表达受体)。
    d。 时间:120秒读取,间隔2秒(或相应选择)
    e。 测定板类型:96孔Greiner blk/clr btm
    6)井读数:整板(或相应选择)
    F。 复合转移:
    • 初始体积:200μl
    • 传输:1(或相应地选择多次传输)
    • 移液器高度:220μl(调整略大于初始体积)
    • 移液器体积:50μl(以使测定板中的配体的最终体积为1x)
    • 评分:1
    • 时间点:18秒(或相应选择)
    G。 化合物来源:Costar 96 well Ubtm clr。 3 ml
    H。 自动校准:在
    上 一世。 自动阅读:关闭
  4. 读取

代表数据



图1.在18℃第一次用(良好H1)和不用(孔A1)1μMSar 1 - 血管紧张素II刺激的HEK293-AT1R细胞。 强>信号随时间的减少很可能是由受体内在化引起的

食谱

  1. 用10%胎牛血清(FBS)提供的细胞生长培养基(对于稳定表达血管紧张素II 1型受体(AT1R)的HEK293细胞)Dulbecco改良的Eagle培养基(DMEM)

致谢

这项工作得到国家卫生研究院拨款HL47570和HL115964(Sadashiva Karnik,Ph。D.)和国家研究服务奖HL007914(Hamiyet Unal,Ph。D.)的支持。

参考文献

  1. Unal,H.,Jagannathan,R.,Bhatnagar,A.,Tirupula,K.,Desnoyer,R.and Karnik,S.S。(2013)。 对血管紧张素II 1型受体的细胞外环2中的特定构象变化的突变的长程作用。 J Biol Chem 288(1):540-551
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Unal, H. (2013). Calcium Mobilization Assay to Measure the Activity of Gq-coupled Receptors. Bio-protocol 3(12): e790. DOI: 10.21769/BioProtoc.790.
  2. Unal, H., Jagannathan, R., Bhatnagar, A., Tirupula, K., Desnoyer, R. and Karnik, S. S. (2013). Long range effect of mutations on specific conformational changes in the extracellular loop 2 of angiotensin II type 1 receptor. J Biol Chem 288(1): 540-551.
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