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cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer)
采用AlphaScreen® 试剂盒(PerkinElmer)进行cAMP累积试验   

编审
Cheng Zhang Cheng Zhang
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Abstract

Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated Gs protein-coupled receptors. Conversely, formation of cAMP is inhibited by Gi protein-coupled receptors. This protocol has been optimized for the detection of ligand-mediated cAMP accumulation in adherent immortal cell lines expressing Gs-coupled receptors.

Materials and Reagents

  1. Sterile 96-well clear flat bottom plates (BD Biosciences, Falcon®, catalog number: 353072 )
  2. Phenol Red Free Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies, Gibco®, catalog number: 21063-029 )
  3. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  4. 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, catalog number: I5879 )
  5. Tween-20 (Sigma-Aldrich, catalog number: P2287 )
  6. HEPES (Life Technologies, Gibco®, catalog number: 11344-041 )
  7. 100% Ethanol
  8. Milli-Q H2O
  9. Alphascreen® cAMP Assay Kit (PerkinElmer, catalog number: 6760625 )
  10. White OptiPlate-384 well microplate (PerkinElmer, catalog number: 6007290 )
  11. TopSeal (PerkinElmer, catalog number: 6005250 )
  12. Stimulation buffer (see Recipes)
  13. Lysis buffer (see Recipes)
  14. Acceptor buffer (see Recipes)
  15. Donor buffer (see Recipes)

Equipment

  1. Fusion-α plate reader or Envision plate reader with appropriate alphascreen modules (PerkinElmer)
  2. Humidified Incubator
  3. Multichannel Pipettes
  4. Micropipettes
  5. Benchtop centrifuge
  6. Oven
  7. Orbital shake

Procedure

Notes:
1) Cells can either be stably or transiently expressing receptor of interest.
2) All incubations are in a humidified environment at 37 °C, 5% CO2 unless otherwise indicated.


I.   Cell preparation

Seed cells in suitable nutrient media (e.g. DMEM, 10% FBS, no antibiotics) into a sterile 96-well plate and incubate in a humidified environment at 37 °C, 5% CO2 to be ~90% confluent the following day (~24 h).
Note: Optimization for cell number depending on the cell line used will be necessary (we suggest a starting range of 10,000-50,000 cells/well). Recommended density for CHO FlpIN cells is 30,000 cells/ well.


II.  Stimulation

  1. The day following seeding, aspirate nutrient media and replace with 90 μl prewarmed Stimulation buffer.
  2. Incubate in a humidified environment at 37 °C, 5% CO2 for 30 min.
    Note: Do not leave in cells in Stimulation buffer for longer than 2 h prior to stimulation.
  3. Prepare serial dilutions of ligands at 10x final concentration in Stimulation buffer, enough for 10 μl/well, to be performed in duplicate (minimum).
    Note: Concentration range to use will depend on ligand affinity for receptor. For initial tests, select a top concentration 100x Kd of ligand, a buffer only control, and several concentrations between these. The range can then be refined in subsequent experiments.
  4. Prepare 10x appropriate concentration of forskolin in Stimulation buffer.
    Note: This is the internal control for the experiment – forskolin is an activator of adenylate cyclase, enhancing the formation of cAMP. Recommended final concentration of forskolin in a CHO FlpIN cell line is 100 μM.
  5. Following 30 min incubation in stimulation buffer, add 10 μl of 10x prepared ligands to cells, for a total volume of 100 μl, 1x final concentration.
  6. Incubate cells with ligand in a humidified environment at 37 °C, 5% CO2 for 30 min.
    Note: Optimization for assay time will be necessary. Recommended initial stimulation is 30 min.
  7. After 30 min, rapidly remove ligand containing media from cells.
    Note: Depending on the cell type, this may involve flicking or gentle aspiration.
  8. Add 50 μl ice cold 100% ethanol to cells.
  9. Allow ethanol to evaporate at room temperature (RT) or in a 37 °C oven.
    Note: Ensure the ethanol is completely evaporated before proceeding to the next step.
  10. Add 75 μl Lysis buffer.
    Note: Optimization for lysis volume will be necessary, and depends on the cell type, expression level of the receptor and efficiency of coupling to the cAMP pathway. Recommended starting lysis volume in a CHO FlpIN cell line is 75 μl.
  11. Incubate lysates at RT for 5-10 min on an orbital shaker.


III. Detection

  1. In reduced lighting conditions, prepare detection reagents (Acceptor and Donor buffers).
  2. Transfer 10 μl of cell lysate to a 384-well OptiPlate.
  3. Prepare cAMP standard curve in Lysis buffer, enough for 10 μl/well to be performed in duplicate (minimum).
  4. Transfer 10 μl cAMP standard curve to a 384-well OptiPlate.
  5. Briefly centrifuge to draw contents to the bottom of the wells.
  6. In reduced lighting conditions, add 5 μl Acceptor buffer to every well (samples and standard curve).
  7. In reduced lighting conditions, add 15 μl Donor buffer to every well (samples and standard curve) (following preincubation for 30 min).
  8. Seal the plate with TopSeal and wrap in foil.
    Note: Small volumes are subject to evaporation, TopSeal is essential.
  9. Briefly centrifuge to draw contents to the bottom of the wells.
  10. Incubate overnight at RT (8-12 h) in reduced lighting conditions.
  11. Briefly centrifuge to draw contents to the bottom of the wells.
  12. Analyse luminescence on a Fusion-α or Envision plate reader using standard α-screen settings.


IV. Data analysis

  1. Extrapolate data from the cAMP standard curve. Ideally, data should lie on the linear section of the cAMP standard curve.
    1. If data falls off the bottom end of the curve (i.e., high concentration of cAMP), dilute lysates further with Lysis buffer and repeat detection component of protocol.
    2. If data falls off the top end of the curve (i.e., low concentration of cAMP), assay should be performed again and cells lysed with a lower volume of Lysis buffer.
  2. Normalize data to forskolin control.

Recipes

  1. Stimulation buffer (pH 7.4, incubate at 37 °C prior to use)
    Phenol free DMEM
    0.1% w/v BSA*
    1 mM 3-isobutyl-1-methylxanthine (IBMX)**
    * BSA is not essential, but is recommended for ‘sticky’ ligands, i.e. peptides.
    ** IBMX is a potent phosphodiesterase inhibitor. IBMX powder should be made up as
    a 500 mM stock in 100% DMSO. It may precipitate out of solution if DMEM is too cold, so gently heat and stir DMEM when adding IBMX.
  2. Lysis buffer (pH 7.4)
    Milli-Q H2O
    0.3% Tween 20
    5 mM HEPES
    0.1% w/v BSA
  3. Acceptor buffer (prepare in Stimulation buffer)
    1% Acceptor beads (10 U/μl) (mix gently by pipetting before use)
  4. Donor buffer (prepare in Stimulation buffer)***
    0.3% Donor Beads (10 U/μl) (mix gently by pipetting before use)
    0.025% biotinylated cAMP (133 U/μl)
    *** Donor buffer MUST be preincubated at RT for 30 min prior to use.
  5. cAMP standard curve
    Prepare cAMP standard dilution series in Lysis buffer at 3x concentration to take into account dilution in detection plate (10 μl cAMP standard, 5 μl Acceptor buffer and 15 μl Donor buffer, final volume 30 μl). Recommended concentration range for cAMP standard (final) is 10 μM - 1 pM in half log units.
    Note: Lysates may be stored at -20 °C and cAMP accumulation can be detected at a later time, but no longer than 2 weeks following stimulation.

References

  1. Koole, C., Wootten, D., Simms, J., Valant, C., Sridhar, R., Woodman, O. L., Miller, L. J., Summers, R. J., Christopoulos, A. and Sexton, P. M. (2010). Allosteric ligands of the glucagon-like peptide 1 receptor (GLP-1R) differentially modulate endogenous and exogenous peptide responses in a pathway-selective manner: implications for drug screening. Mol Pharmacol 78(3): 456-465.

简介

环腺苷一磷酸(cAMP)是来源于ATP的催化转化的细胞内信号传导信号,并且是活化的G蛋白偶联受体的主要产物。 相反,cAMP的形成受G 1 i蛋白偶联受体抑制。 该方案已经被优化用于检测表达G s - 偶联受体的贴壁无限增殖细胞系中配体介导的cAMP积累。

材料和试剂

  1. 无菌96孔透明平底板(BD Biosciences,Falcon ,目录号:353072)
  2. 酚红免费Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,Gibco ,目录号:21063-029)
  3. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  4. 3-异丁基-1-甲基黄嘌呤(IBMX)(Sigma-Aldrich,目录号:I5879)
  5. 吐温-20(Sigma-Aldrich,目录号:P2287)
  6. HEPES(Life Technologies,Gibco ,目录号:11344-041)
  7. 100%乙醇
  8. Milli-Q H sub 2 O 2 /
  9. Alphascreen cAMP测定试剂盒(PerkinElmer,目录号:6760625)
  10. White OptiPlate-384孔微孔板(PerkinElmer,目录号:6007290)
  11. TopSeal(PerkinElmer,目录号:6005250)
  12. 刺激缓冲区(参见配方)
  13. 裂解缓冲液(见配方)
  14. 接受者缓冲区(参见配方)
  15. 捐助者缓冲区(参见配方)

设备

  1. Fusion-α板读取器或Envision读板器和适当的alphascreen模块(PerkinElmer)
  2. 加湿孵化器
  3. 多通道移液器
  4. 微量移液器
  5. 台式离心机
  6. 烤箱
  7. 轨道抖动

程序

注意:
1)细胞可以稳定或瞬时表达感兴趣的受体。
/sub> ,除非另有说明。


I.   细胞制备

在合适的营养培养基(例如DMEM,10%FBS,无抗生素)中将细胞接种到无菌96孔板中,并在37℃,5%CO 2的潮湿环境中培养, /约为〜90%,第二天融合(约24小时)。
注意:根据所使用的细胞系对细胞数目的优化是必要的(我们建议起始范围为10,000-50,000个细胞/孔)。 CHO FlpIN细胞的推荐密度为30,000个细胞/孔


II。  刺激

  1. 播种后的一天,吸入营养培养基并更换为90μl预热的刺激缓冲液
  2. 在37℃,5%CO 2的潮湿环境中孵育30分钟。
    注意:刺激前不要在刺激缓冲液中离开细胞超过2小时。
  3. 准备在刺激缓冲液中10×终浓度的配体的系列稀释液,以足够10μl/孔,一式两份(最小)。
    注意:使用的浓度范围将取决于配体对受体的亲和力。 对于初始测试,选择最高浓度100×K d配体,仅缓冲液对照,以及这些之间的几个浓度。 然后可以在后续实验中细化范围。
  4. 准备10倍适当浓度的毛喉素在刺激缓冲液中。
    注意:这是实验的内部对照 - forskolin是腺苷酸环化酶的激活剂,增强cAMP的形成。 在CHO FlpIN细胞系中推荐的毛喉素的最终浓度为100μM。
  5. 在刺激缓冲液中孵育30分钟后,向细胞中加入10μl10x制备的配体,总体积为100μl,最终浓度为1x。
  6. 在37℃,5%CO 2的潮湿环境中与配体孵育细胞30分钟。
    注意:需要优化测定时间。 推荐的初始刺激为30分钟。
  7. 30分钟后,从细胞中快速除去含配体的培养基。
    注意:根据细胞类型,这可能涉及轻拂或轻柔吸气。
  8. 加入50微升冰冷的100%乙醇的细胞。
  9. 让乙醇在室温(RT)或37℃烘箱中蒸发 注意:确保乙醇完全蒸发,然后再进行下一步骤。
  10. 加入75μl裂解缓冲液。
    注意:裂解体积的优化是必要的,并且取决于细胞类型,受体的表达水平和偶联到cAMP途径的效率。 在CHO FlpIN细胞系中推荐的起始裂解体积为75μl。
  11. 孵育裂解物在室温下5-10分钟在轨道摇床


III。 检测

  1. 在减光条件下,准备检测试剂(受体和供体缓冲液)
  2. 转移10微升细胞裂解物到384孔OptiPlate。
  3. 在裂解缓冲液中制备cAMP标准曲线,足以以10μl/孔重复(最小)进行
  4. 转移10微升cAMP标准曲线到384孔OptiPlate
  5. 短暂离心以将内容物吸到孔底部
  6. 在减光条件下,向每个孔中加入5μl受体缓冲液(样品和标准曲线)
  7. 在减少照明条件下,向每个孔(样品和标准曲线)中加入15μl供体缓冲液(预温育30分钟后)。
  8. 用TopSeal密封板并用箔包裹。
    注意:小体积容易蒸发,TopSeal是至关重要的。
  9. 短暂离心以将内容物吸到孔底部
  10. 在室温(8-12小时)在减光条件下孵育过夜
  11. 短暂离心以将内容物吸到孔底部
  12. 使用标准α屏幕设置在Fusion-α或Envision读板仪上分析发光


IV。 数据分析

  1. 从cAMP标准曲线推断数据。 理想情况下,数据应位于cAMP标准曲线的线性部分。
    1. 如果数据从曲线的底端(即,cAMP的高浓度)下降,用裂解缓冲液进一步稀释裂解物,并且方案的重复检测组分。
    2. 如果数据从曲线的顶端脱落( i ,cAMP浓度低) 用较低体积的裂解缓冲液裂解。
  2. 将数据规范化为forskolin控制。

食谱

  1. 刺激缓冲液(pH 7.4,使用前在37℃下孵育)
    无酚DMEM
    0.1%w/v BSA *
    * BSA不是必需的,但推荐用于"粘性"配体,即肽。
    ** IBMX是一种有效的磷酸二酯酶抑制剂。 IBMX粉末应该是
    在100%DMSO中的500mM储备液。 如果DMEM太冷,可能从溶液中沉淀出来,因此在添加IBMX时轻轻加热并搅拌DMEM
  2. 裂解缓冲液(pH 7.4)
    Milli-Q H sub 2 O 2/0.3%Tween 20
    5 mM HEPES
    0.1%w/v BSA
  3. 受体缓冲液(在刺激缓冲液中制备)
    1%受体珠(10 U /μl)(使用前用移液管轻轻混匀)
  4. 供体缓冲液(在刺激缓冲液中制备)***
    0.3%供体珠(10U /μl)(使用前通过移液轻轻混合)
    0.025%生物素化的cAMP(133U /μl) ***供体缓冲液在使用前必须在RT预温育30分钟
  5. cAMP标准曲线
    准备在3x浓度的裂解缓冲液中的cAMP标准稀释系列,以考虑检测板中的稀释(10μlcAMP标准品,5μl受体缓冲液和15μl供体缓冲液,终体积30μl)。 cAMP标准品(最终)的推荐浓度范围为10μM - 1 pM,以半对数单位表示。
    注意:裂解物可以储存在-20°C,cAMP累积可以稍后检测,但刺激后不超过2周。

参考文献

  1. Koole,C.,Wootten,D.,Simms,J.,Valant,C.,Sridhar,R.,Woodman,O.L.,Miller,L.J.,Summers,R.J.,Christopoulos,A.and Sexton,P.M。 胰高血糖素样肽1受体(GLP-1R)的变构配体差异调节内源和外源肽 response in a pathway-selective manner:implications for drug screening。 Mol Pharmacol 78(3):456-465。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Koole, C., Wootten, D. and Sexton, P. M. (2013). cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer) . Bio-protocol 3(13): e805. DOI: 10.21769/BioProtoc.805.
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