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Introduction

FLIPR^®Calcium Assay Kits from Molecular Devices employ sensitive calcium indicators and proprietary masking dyes to enable researchers to conduct highly sensitive fluorescent screens of G-protein coupled receptors (GPCRs), ion channels, and other calcium sensitive targets. By using a novel dye formulation to further enhance the calcium flux assay signal window, assay robustness is increased, while providing greater assay protocol flexibility. As a result, the FLIPR Calcium6 and Calcium 6-QF Calcium Kit dyes become more suitable for measuring calcium flux in 384-well plates with the medium throughput FlexStation^®3 MultiMode Microplate Reader using the 16 channel pipettor. Results are similar to those obtained on the FLIPR^®Tetra System in high throughput mode.

Assay principle

As shown in Figure 1, the FLIPR Calcium 6 assay dye enters the cytosol of the cell. The masking technology does not enter the cell but significantly reduces background fluorescence originating from residual extracellular calcium indicator, media, and other components. The FLIPR Calcium 6-QF Assay Kit formulation contains no masking technology and delivers a new, flexible option for quench sensitive targets or multiplexing applications. Additional assay flexibility is provided with minimal to no requirement for use of probenecid in the assays. Certain cells such as Chinese Hamster Ovary (CHO) cell lines have an anion-exchange protein that requires the use of an anion reuptake inhibitor, such as probenecid, to retain commonly used calcium indicators within the cytosol. The unique FLIPR Calcium6 Assay Kit dye formulation is more resistant to such organic anion transporters, thus less or no probenecid may be required.

Figure 1. Assay flexibility with or without masking technology.

Materials and methods

An assay examining the endogenous histamine receptor expressed in HeLa cells was developed using the new FLIPR Calcium 6 and Calcium 6-QF Assay Kits, and then compared to other commercially available kits. Calcium flux was measured with the FlexStation 3reader using the ‘Flex’ read mode. HeLa cells kept in continual culture were plated at 5,000 cells/well in 50 µL growth media in blackwall, clear bottom 384-well microplates and then maintained overnight at 37°C, 95% humidity, and 5% CO₂. On the following day, cell plates were loaded with the appropriate calcium kit reagents following manufacturers’ recommendations (including water soluble probenecid) and incubated for 60 or 120 minutes according to manufacturer protocol.

The cells were challenged with varying concentrations of histamine (starting at 100 µM, in 3-fold dilutions) using the FlexStation 3 reader’s integrated 16-channel pipettor. Fluorescence measurements were taken for 60 seconds before, during, and after compound addition using optimized parameters (Table 1.) For the antagonist studies, pyrilamine and risperidone (starting at 30 µM, in 3-fold dilutions) were added using the FlexStation 3 reader’s on-board fluidic system and allowed to equilibrate for 30 minutes. The cells were then stimulated with 15 µL/well of histamine (EC₈₀concentration) while changes in fluorescence intensity were monitored in real time.

The calcium indicators used for the comparison were:

• FLIPR Calcium6 Assay Kit (Molecular Devices)
• FLIPR Calcium 6-QF Assay Kit (Molecular Devices)
• Fluo-4 Direct Calcium Assay Kit (Life Technologies)
• Fluo-4 Direct Calcium Assay Kit (Life Technologies)

Table 1. FlexStation reader settings. Optimized instrument settings for the calcium assays described are shown.

FlexStation 3 reader settings

Cell plates were maintained at 37°C inside the FlexStation 3 reader. Compounds were prepared in Greiner 384-well polystyrene deep-well plates and FlexStation 3 Tips (black) were used for compound transfer. The specific parameters are indicated in Table 1.

Results and analysis

Responses were measured as peak fluorescence intensity. To enable comparison, data was normalized as % response over baseline and expressed as mean ± standard deviation with n=4. Individual sets of concentration-response data were fitted to a four-parameter curve using SoftMax^®Pro Software (Figures 2-5).

Figure 2. FLIPR Calcium 6 and 6-QF Assay Kits provide the largest signal window. Histamine H1 is an endogenous receptor in HeLa cells. Comparing FLIPR Calcium 6 and 6-QF to other dyes shows that both had the largest signal window. EC₈₀values were within one-half log and Z factors at EC₈₀were comparable.

Figure 3. Risperidone antagonist response to histamine challenge in HeLa cells. Risperidone is an antipsychotic drug used to treat schizophrenia. It is a dopamine antagonist that also has antihistamine properties. The signal from both Calcium 6 and 6-QF dyes provides the largest window for the antagonist assay. IC₅₀values are within one-half log of each other and Calcium 6-QF has the highest Z factor at IC₅₀concentration.

Figure 4. Pyrilamine antagonist response to histamine challenge in HeLa cells. Pyrilamine is a first generation Histamine H1 antagonist. Because of the larger signal window provided by the FLIPR Calcium 6 Assay Kit, the Z factor at IC₅₀is the largest. In addition, the Calcium 6-QF kit also provides a robust assay that does not require washing when quench sensitive targets are to be studied.

Figure 5. FLIPR Calcium 6 assay dye does not require use of anion reuptake inhibitors. Assay performed with CHO-M1 cells and Calcium 6 dye demonstrates a response to carbachol without the need for incubation with probenecid. The corresponding assay with Fluo-4 Direct shows virtually no signal. Calcium 6 dye is an important new development for understanding targets that may be sensitive to anion reuptake inhibitors.

Conclusion

FLIPR Calcium6 and Calcium 6-QF Assay Kits offer flexible calcium flux assays including medium throughput options such as the FlexStation 3 reader while providing reliable pharmacology, a superior signal window, and high quality assay performance. Having a greater signal window is important because many of today’s assays present challenges not seen with standard agonist or antagonist assays using over-expressed receptors. Use of cell lines with endogenous receptors, lower expression of receptors, frozen cells, primary cells, or stem cells may produce lower signal windows. In addition, a larger signal window is an advantage when performing assays to identify allosteric modulators.

The FLIPR Calcium 6-QF Assay Kit option without quench provides new assay flexibility to enable assays for studying quench sensitive targets. Lastly, the ability to study target behavior in cell lines such as CHO cells in the absence of an anion reuptake inhibitor can be beneficial as some receptors and ion channelsmay be probenecid sensitive and its use may alter the natural biological mechanisms. The larger signal window provided by FLIPR Calcium 6 Kits delivers a robust assay for compound screening and optimization utilizing these challenging targets and cell lines.

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简介

来 自 Molecular Devices 公 司 的 FLIPR® Calcium 检测试剂盒使用敏感的钙指示剂 和专有的掩蔽染料，使研究人员能够对 G 蛋白偶联受体 (GPCRs)、离子通道和其他 钙敏感靶点进行高度敏感的荧光筛选。通 过使用一种新的染料配方来进一步增强钙 流检测信号窗口，提高了测定的稳定性，同 时提供了更高的测定方案灵活性。因此， FLIPR Calcium 6 和 Calcium 6-QF 钙试剂 盒染料更适合于使用 16 通道移液头以中等 通量的 FlexStation® 3 多功能微孔板读板 机测量 384 孔板中的钙流。结果与 FLIPR® Tetra 系统在高通量模式下获得的结果相 似

检测原理

如 图 1 所 示 , FLIPR Calcium 6 检 测 染 料 进入细胞的胞质中。掩蔽技术不进入细胞， 但显著减少了来自残留细胞外的钙指示 剂、培养基和其他成分的背景荧光。FLIPR Calcium 6-QF 检测试剂盒配方不含掩蔽技 术，为淬灭敏感的靶标或多色应用提供了 一种新的、灵活的选择。提供了额外的检测 灵活性，在检测中使用丙磺舒的要求最小 到没有要求。某些细胞，如中国仓鼠卵巢 (CHO) 细胞系中有一种阴离子交换蛋白， 需要使用阴离子再摄取抑制剂，如丙磺舒， 才能在胞质中保留常用的钙指示剂。独特 的 FLIPR Calcium 6 检测试剂盒染料配方 对此类有机阴离子转运体具有更强的抗性， 因此可能需要较少或不需要丙磺舒。

图 1 具有或不具有掩蔽技术的检测灵活性。

材料和方法

使用新的 FLIPR Calcium 6 和 Calcium 6-QF 检测试剂盒开发了检测 HeLa 细胞中表达 的内源性组胺受体的方法，然后与其他商 业上可用的试剂盒进行比较。采用‘Flex’ 读数模式在 FlexStation 3 读板机上测定钙 流。将保存在连续培养中的 HeLa 细胞以 5000 个细胞 / 孔接种在含有 50 µL 生长培 养基的黑壁、底透的 384 孔微孔板中，然后 在 37℃、95% 湿度和 5% CO2 下过夜。第 二天，细胞培养板按照制造商的建议 ( 包括 水溶性丙磺舒 ) 加入适当的钙试剂盒试剂， 并根据制造商的方案孵育 60 或 120 分钟。

使用 FlexStation 3 读板机集成的 16 通道 移液器，用不同浓度的组胺 ( 从 100 µM 开 始， 3 倍稀释 ) 对细胞提出挑战。使用优化 的参数在化合物添加前中后进行 60 秒的 荧光测定 ( 表 1 )。在拮抗剂研究中，使用 FlexStation 3 读 板 机 的 机 载 移 液 系 统 加 入 嘧 啶 和 利 培 酮 ( 从 30 µM 开 始，3 倍 稀 释 )，并允许平衡 30 分钟。然后用 15 µL/ 孔的组胺 ( EC80 浓度 ) 刺激细胞，同时实时 监测荧光强度的变化。

比较所用的钙指标是：

• FLIPR Calcium 6 检测试剂盒 (Molecular Devices)
• FLIPR Calcium 6-QF 检测试剂盒 (Molecular Devices)
• Fluo-4 Direct Calcium 检测试剂盒 (Life Technologies)

***表 1 FlexStation 读板机设置。*结合 Opti-MEM 培养基、DNA 和转染试剂在本体系反应 中形成转染复合物。制备足够的复合物，为每个实验条件转染 10 个孔。

FlexStation 3 读板机设置

细 胞 板 在 FlexStation 3 读 板 机 中 维 持 37℃。将化合物准备在 Greiner 384 孔聚 苯乙烯深孔板中，用 FlexStation 3 移液头 ( 黑色 ) 进行化合物转移。具体参数见表 1。

结果和分析

以峰值荧光强度测定响应。为了便于比较， 将数据归一化为基线上的 % 响应，并表示 为 n=4 的平均 ± 标准差。使用 SoftMax® Pro 软件将各组浓度响应数据拟合到四参 数曲线上 ( 图 2-5 )。

***图 2 FLIPR Calcium 6 和 6-QF 检测试剂盒提供最大的信号窗口。*组胺 H1 是 HeLa 细胞 的内源性受体。将 FLIPR Calcium 6 和 6-QF 与其他染料进行比较，发现两者都有最大的 信号窗口。EC80 值在一半对数和 Z 因子在 EC80 处具有可比性。

***图 3 利培酮拮抗剂对 HeLa 细胞组胺作用的响应。*利培酮是一种治疗精神分裂症的抗精 神病药物。它是一种多巴胺拮抗剂，也具有抗组胺作用。Calcium 6 和 6-QF 染料的信号 为拮抗剂测定提供了最大的窗口。在 IC50 浓度下，IC50 值在一半对数和 Calcium 6-QF 的 Z′ 因子最高。

***图 4 嘧啶胺拮抗剂对 HeLa 细胞组胺作用的响应。*嘧啶胺是第一代组胺 H1 拮抗剂。 由于 FLIPR Calcium 6 检测试剂盒提供的信号窗口较大，IC50 处的 Z′因子最大。此外， Calcium 6-QF 试剂盒还提供了一种可靠的检测方法，在研究淬灭敏感靶标时不需要洗涤。

***图 5 FLIPR Calcium 6 检测染料不需要使用阴离子再摄取抑制剂。*用 CHO-M1 细胞和 Calcium 6 染料进行的测定显示了对碳酰胆碱的响应，而不需要与丙磺舒孵育。相对应的 Fluo-4 Direct 检测几乎没有信号。Calcium 6 染料是了解可能对阴离子再摄取抑制剂敏感 的靶点的重要新进展。

总结

FLIPR Calcium 6 和 Calcium 6-QF 检测试 剂盒提供灵活的钙流检测，包括中等通量 选项，如 FlexStation 3 读板机，同时提供 可靠的药理学、优越的信号窗口和高质量 的检测性能。获得更大的信号窗口是很重 要的，因为现在的许多实验都面临着挑战， 已见不到标准的激动剂或拮抗剂使用过表 达的受体进行实验了。使用内源性受体、受 体低表达、冷冻细胞、原代细胞或干细胞的 细胞系可能产生较低的信号窗口。此外，更 大的信号窗口在进行实验以识别变构调节 剂方面是一个优势。

不会淬灭的 FLIPR Calcium 6-QF 检测试剂 盒选项为研究淬灭敏感靶点提供了新的检 测灵活性。最后，在没有阴离子再摄取抑制 剂的情况下，研究诸如 CHO 细胞系中靶行 为的能力是有益的，因为某些受体和离子 通道可能是丙磺舒敏感的，其使用可能会 改变自然的生物学机制。由 FLIPR Calcium 6 试剂盒提供的更大的信号窗口使其成为 一个强有力的检测方法，用于利用这些具 有挑战性的靶标和细胞系进行化合物筛选 和优化。

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