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Introduction

In this study, we demonstrate how the CatchPoint^®cAMP Fluorescent Assay Kitcombined with the SpectraMax^®i3 MultiMode Detection Platform can be used to monitor the response of HEK293 cells to forskolin, an activator of adenylate cyclase (Figure 1).

Figure 1: CatchPoint cAMP workflow

G-Protein Coupled Receptors (GPCRs) are important transmembrane proteins that translate extracellular signals into intracellular responses. These intracellular responses are comprised of cell-signaling cascades that initiate changes in protein activity and expression within the cell [1].

Cyclic adenosine 3’, 5’-monophosphate (cAMP) is a secondary messenger that works downstream of GPCRactivation. Upon ligand binding to a GPCR, a conformational change occurs, activating the receptor and in turn activating a G protein. Further signal transduction depends on the type of G protein activated. Activation of G_sleads to upregulation of cAMP by adenylate cyclase, which activates protein kinase A, causing phosphorylation of targets involved in processes such as dopamine signaling, gluconeogenesis, vasodilation, as well as mitogenesis and oocyte maturation [2–6].

The CatchPoint cAMP Fluorescent Assay Kit measures cAMP levels via a competitive immunoassay (Figure 2). The assay requires only a single washing step, and readings can be taken in as little as 10 minutes or as long as 24 hours following substrate addition.

Figure 2: CatchPoint cAMP assaymechanism. Unlabeled cAMP produced by cells competes with cAMP-HRP conjugate for binding to anti-cAMP antibodies. Increasing amounts of cellular cAMP are detected through decreasing HRP activity.

Materials

• CatchPoint cAMP Fluorescent Assay Kit(Molecular Devices cat. #R8088)
• HEK293 cells (ATCC cat. #CRL-1573)
• Krebs-Ringer bicarbonate buffer - KRGB buffer (Sigma cat. #K4002)
  • Sodium bicarbonate (Sigma, cat. #S5761)
• Phosphate-buffered saline (PBS, Life Technologies cat. #10010)
• Phosphodiesterase inhibitor, 3-isobutyl-1- methylxanthine (IBMX, Sigma cat. #I7018)
• 3% hydrogen peroxide (H ₂ 0 ₂ ) solution
• Minimum Essential Medium with Earle’s salts and L-glutamine (Corning cat. #10-010)
  • Fetal bovine serum (Gemini Bio-Products cat. #100-106)
  • Penicillin-streptomycin (Life Technologies cat. #15070-063)
  • Forskolin (Sigma cat. #F6886)
  • Poly-D-Lysine Coated 96-well Microplates (Corning cat. #354413)
  • SpectraMax i3 Multi-Mode Microplate Reader
  • SpectraMax ^® MiniMax ^™ 300 Imaging Cytometer
  • MultiWash+ ^™ Microplate Washer

Methods

HEK293 cells were cultured in complete growth medium (MEM + 10% FBS + 1% Pen/ Strep) and grown to 80-90% confluence in T75 flasks. Cells were harvested using 0.05% trypsin, and 25,000 cells were seeded in each well of a poly-D-lysine coated, 96-well, black-wall, clear-bottom microplate (25,000-100,000 cells/well is an acceptable range). Cells were allowed to adhere for at least 18 hours in a 37°C incubator with 5% CO₂.

On the day of assay, cell confluence was measured using the transmitted light channel of the SpectraMax MiniMax 300 Imaging Cytometerand StainFree^™Cell Detection Technology, which enables accurate measurement of cell counts or percent area covered by cells (confluence) for quality control purposes (Figure 3). Rows of cells with high cell confluence and low inter-well variability were selected for the CatchPoint assay. Cells were pre-stimulated with 0.75 mM IBMX for 10 minutes at room temperature. They were then treated with a dilution series of forskolin starting at 1,000 µM with a 1:3 dilution series for 15 minutes at 37°C. Cells were then lysed according to the CatchPoint assay protocol.

Figure 3: HEK293 cell percent confluence determined using the MiniMax cytometer and StainFree analysis. Rows A and C had similar cell confluence and the lowest variability between wells and were selected for assay.

The CatchPoint assay was performed as described in the product insert. A cAMP calibrator curve was performed to verify assay performance, as well as provide a means of calculating the amount of cAMP in cellular assay samples. Washing was performed on the MultiWash+ Microplate Washer. Samples were read on a SpectraMax i3 Multi-Mode Microplate Reader 30 minutes after StopLight Red substrate addition. All data analysis and curve fitting were performed with SoftMax^®Pro Software. A preconfigured protocol is available in the software’s protocol library.

Results

To assess cell confluence before stimulation, the MiniMax cytometer and StainFree technology were used to measure percent cell coverage in wells of the microplate (Figure 3). Rows A and C were chosen for the assay due to consistent percent confluence from well to well. Triplicate samples were taken from each treated well and assayed.

Figures 4 and 5 show results for the CatchPoint cAMP calibrator curve and cell-based assay. Both curves were generated using the 4-parameter curve fit in SoftMax Pro Software. The calibrator curve had an EC₅₀of 3.3 nM, which agreed closely with previous published data [7]. The Z’ factor was 0.91.

Figure 4: cAMP calibrator curve. EC₅₀was 3.3 nM, similar to previously published results. Samples were run in duplicate.

Figure 5: Cell-based assay performed with HEK293 cells. A forskolin concentration-response curve was run, starting at 1,000 µM with a 3-fold dilution series, with samples run in duplicate. EC₅₀was 2.3 µM.

The forskolin concentration-response curve had an EC₅₀of 2.3 µM, which is consistent with expected results for HEK293 cells assayed in an adherent format

Conclusion

The CatchPoint cAMP FluorescentAssay Kit allows for accurate measurements of GPCRactivity through cAMP detection. High signal stability (10 minutes to 24 hours), excellent Z’ factor, and the ability to be performed on plate readers with a fluorescence intensity detection mode make the CatchPoint assay a versatile choice for high-throughput screening.

Compatible with these Molecular Devices systems

References

1. Zaccolo, Manuela. “cAMP signal transduction in the heart: understanding spatial control for the development of novel therapeutic strategies.” British Journal of Pharmacology 158.1 (2009): 50-60.
2. Hanoune, Jacques, and Nicole Defer. “Regulation and role of adenylyl cyclase isoforms.” Annual Review of Pharmacology and Toxicology 41.1 (2001): 145-174.
3. Griendling, Kathy K., et al. “Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.” Arteriosclerosis, Thrombosis, and Vascular Biology 20.10 (2000): 2175-2183.
4. Kemp, Bruce E., et al. “Dealing with energy demand: the AMP-activated protein kinase.” Trends in Biochemical Sciences 24.1 (1999): 22-25.
5. Etgen, Anne M., Michael A. Ansonoff, and Arnulfo Quesada. “Mechanisms of ovarian steroid regulation of norepinephrine receptor-mediated signal transduction in the hypothalamus: implications for female reproductive physiology.” Hormones and Behavior 40.2 (2001): 169-177.
6. Chini, Eduardo N., et al. “Adrenomedullin suppresses mitogenesis in rat mesangial cells via cAMP pathway.” Biochemical and Biophysical Research Communications 215.3 (1995): 868-873.
7. Hesley, Jayne, Janet Daijo, and Anne T. Ferguson. “Stable, sensitive, fluorescence-based method for detecting cAMP.” BioTechniques 33.3 (2002): 692-694.

Learn more about EarlyTox Cardiotoxicity Kit >>

介绍

本研究展示了如何在 SpectraMax® i3多功 能酶标仪中应用CatchPoint® cAMP荧光 试剂盒监测腺苷酸环化酶激活剂forskolin 对HEK293细胞的影响(如图1)。

图1：CatchPoint cAMP检测流程

G蛋白偶联受体(GPCRs)是重要的跨膜蛋 白，能将胞外信号传导入胞内，引起信号 逐级放大的级联反应，导致胞内某些蛋白 活性或表达的改变[1]。

3’, 5’-单磷酸化环 腺苷(cAMP)作为第二信使参与GPCR活 化后的下游反应。当胞外配体作用于 GPCR时，GPCR构象发生改变，激活胞 内连接的G蛋白。接下来的信号传导路径 与被激活的G蛋白类型有关。其中当Gs蛋 白被激活时，会导致腺苷酸环化酶活化引 起的胞内cAMP浓度上调，进一步激活蛋 白激酶A，最终促进一些目标蛋白的磷酸 化，这些目标蛋白可能参与了例如多巴胺 神经信号传导、葡萄糖再生反应、血管扩 张、细胞有丝分裂、卵子成熟等重要生理 生化过程[2-6]。

CatchPoint® cAMP荧光检测试剂盒采用 免疫竞争原理测得cAMP浓度(如图2)，仅 需一步洗脱，且反应底物加入后短可放置 10分钟，长可延至24小时后再检测，信号 稳定灵敏。

图2：CatchPoint cAMP检测原理 细胞中含有的未标记cAMP与试剂盒中提供的HRP标记cAMP竞 争结合上孔板包被的cAMP抗体。通过检测HRP活性降低可反映细胞内cAMP含量增多。

材料

• CatchPoint cAMP荧光检测试剂盒 (Molecular Devices ，货号R8088)
• HEK293细胞 (ATCC 编号CRL-1573)
• KRGB缓冲液 (Sigma，货号K4002)
  • 碳酸氢钠 (Sigma，货号S5761)
• PBS缓冲液 (Life Technologies， 货号10010)
• 磷酸二酯酶抑制剂，3-异丁基-1-甲基黄嘌呤， IBMX (Sigma，货号17018)
• 30%过氧化氢溶液
• MEM培养基 (Corning，货号10-010)
  • 胎牛血清 (Gemini Bio-Products，货号100-106)
  • 青霉素 (Life Technologies，货号15070-063)
  • Forskolin (Sigma，货号F6886)
  • 多聚-D-赖氨酸包被96孔板 (Corning，货号354413)
  • SpectraMax® i3多功能酶标仪
  • SpectraMax® MiniMax™ 300细胞成像系统
  • • MultiWash+™ 洗板机

方法

HEK293细胞培养于全生长培养基 (MEM+10%FBS+1%青霉素/链霉素) 中，铺满T75培养瓶瓶底80-90%。 0.05%胰酶消化后收集细胞，按照25000 个/孔的密度接种到96孔板中(多聚-D-赖 氨酸包被，黑壁底透)，接种密度可在 25000-100000个/孔范围，37°C、 5%CO2环境下细胞贴壁培养至少18h。

荧光检测前，先用SpectraMax® MiniMax™ 300细胞成像系统透射光通道观察细胞， 以掌握其生长饱和情况，并应用StainFree™无标记细胞计数法准确测量细胞数 目或覆盖面积百分比(即饱和度)，以达到 质量控制的目的(如图3)。细胞饱和度高且 孔间差异小的行被挑选进行CatchPoint检 测。室温下先用0.75mM IBMX预作用细 胞10分钟，然后37°C下用系列浓度梯度 的forskolin(起始孔浓度1000nM，其他孔 按1:3依次稀释)处理细胞15分钟。药物处 理后按照CatchPoint检测试剂盒说明书裂 解各孔细胞。

图3：利用MiniMax细胞成像系统和无标记技术检测HEK293细胞饱和度 A行和C行具有相似的细胞 饱和度百分比以及最小的孔间差异，因此被选中进行检测

接下来按照产品说明书进行操作检测。 cAMP标准样品检测得到一条标准曲线， 以确认试剂盒功能是否正常，并可通过该 拟合方程式计算出细胞样品中cAMP浓 度。过程中洗脱步骤使用MultiWash+洗 板机。红色底物StopLight加入样品30分 钟后用SpectraMax® i3多功能酶标仪读数。 SoftMax® Pro软件执行所有数据采集、分 析和曲线拟合功能，另外还在模板库中提 供了专门的CatchPoint cAMP检测试剂 盒预设模板。

结果

在药物刺激前需掌握细胞饱和度情况，该 实验中采用了Minimax成像系统以及 StainFree无标记技术检测各孔中细胞覆 盖面积百分比(如图3)。根据检测结果，A 行和C行因为不同孔间均一的细胞生长饱 和度被选作待测样品行。各处理孔提取出 细胞裂解后的样品进行检测。

图4和5分别显示了cAMP标准曲线和细胞 样品药物浓度响应曲线，两条曲线均用4 参数拟合。标准曲线得到EC50=3.3 nM， 该值与已发表数据一致[7]，其中Z因子为 0.91。

图4：cAMP标准曲线 EC50 为3.3 nM，该值与已发表数据一致，样品检测2个复孔

图5：HEK293细胞样品检测结果 得到一条forskolin浓度响应曲线，从起始浓度1000nM开始按1/3 稀释的浓度梯度，每种处理样品检测2个复孔，EC50 为2.3 nM

Forskolin浓度响应曲线测得EC50=2.3 nM，该值符合根据HEK293细胞贴 壁情况推导的结论。

结论

CatchPoint® cAMP荧光检测试剂盒能通 过测量cAMP浓度准确衡量GPCR活性， 检测信号极稳定(可维持10分钟-24小 时)，优异的Z因子值，应用酶标仪荧光强 度功能进行CatchPoint cAMP检测成为 高通量药物筛选的可靠选择。

该实验所使用的Molecular Devices酶标仪：

参考文献

1. Zaccolo, Manuela. “cAMP signal transduction in the heart: understanding spatial control for the development of novel therapeutic strategies.” British Journal of Pharmacology 158.1 (2009): 50-60.
2. Hanoune, Jacques, and Nicole Defer. “Regulation and role of adenylyl cyclase isoforms.” Annual Review of Pharmacology and Toxicology 41.1 (2001): 145-174.
3. Griendling, Kathy K., et al. “Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.” Arteriosclerosis, Thrombosis, and Vascular Biology 20.10 (2000): 2175-2183.
4. Kemp, Bruce E., et al. “Dealing with energy demand: the AMP-activated protein kinase.” Trends in Biochemical Sciences 24.1 (1999): 22-25.
5. Etgen, Anne M., Michael A. Ansonoff, and Arnulfo Quesada. “Mechanisms of ovarian steroid regulation of norepinephrine receptor-mediated signal transduction in the hypothalamus: implications for female reproductive physiology.” Hormones and Behavior 40.2 (2001): 169-177.
6. Chini, Eduardo N., et al. “Adrenomedullin suppresses mitogenesis in rat mesangial cells via cAMP pathway.” Biochemical and Biophysical Research Communications 215.3 (1995): 868-873.
7. Hesley, Jayne, Janet Daijo, and Anne T. Ferguson. “Stable, sensitive, fluorescence-based method for detecting cAMP.” BioTechniques 33.3 (2002): 692-694.

Learn more about EarlyTox Cardiotoxicity Kit >>

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