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Hoang Ha| Applications Scientist | Molecular Devices

Introduction

Reporter gene assays are important tools for studying gene expression associated with the activation of cellular pathways. Cells are transfected with a plasmid containing the reporter gene and a sequence of interest, typically a promoter or other transcriptional control element. When the promoter is activated, the reporter gene is expressed and its levels can be measured.

Firefly luciferase is a widely-used reporter gene. Its luminescent signal offers exquisite sensitivity compared to fluorescence or other methods. A second luminescent reporter, Renilla luciferase, is often used under the control of a constitutive promoter to normalize for sources of variability such as transfection efficiency and cell number. Since firefly and Renilla luciferases use different substrates, both can be measured in the same well using a dual luciferase detection assay

The SpectraMax® DuoLuc Reporter Assay Kitenables highly sensitive quantitation of both firefly and Renilla luciferases in a microplate format. Addition of firefly working solution to a sample well initiates the firefly luminescence reaction, and subsequent addition of Renilla working solution simultaneously quenches the firefly and initiates the Renilla reaction.

The FlexStation® 3 Multi-Mode Microplate Reader can be used to run the SpectraMax DuoLucassay in 96- and 384-well formats with high sensitivity and throughput. On the FlexStation 3 reader, the entire series of reactions is monitored in real time with simultaneous on-board reagent pipetting and luminescence detection (Figure 1).

Materials

• FlexStation 3Multi-Mode Microplate Reader (Molecular Devices cat. #Flex3)
  • FlexStation 3 8-channel Pipettor Head (Molecular Devices cat. #0200-6182)
  • FlexStation 3 16-channel Pipettor Head (Molecular Devices cat. #0200-6183)
  • 96-well Black FlexStation Pipet Tips (Molecular Devices cat. #0900-0911)
  • 384-well Black FlexStation Pipet Tips (Molecular Devices cat. #9000-0764)
• SpectraMax DuoLuc ReporterAssay Kit (Molecular Devices cat. #R8361)
• HeLa cells (ATCC cat. #CCL-2)
• pGL4.13[luc2/SV40] firefly luciferase expression vector (Promega cat. #E668A)
• pGL4.75[hRluc/CMV] Renilla luciferase expression vector (Promega cat. #E693A)
• ViaFect™ Transfection Reagent (Promega cat. #E4981)
• Opti-MEM Reduced Serum Medium (ThermoFisher Scientific cat. #31985062)

Figure 1. The reaction is monitored on the FlexStation 3 reader in real time as reagent additions occur. Area under the curve (AUC) for a specified portion of each phase of the reaction (firefly and Renilla) is calculated by the software.

Methods

Cell Transfection

HeLa cells were seeded at 2x105 cells per well in 6-well tissue-culture treated plates and incubated at 37°C/5% CO₂for 24 hours prior to transfection. The pGL4.13[luc2/SV40] firefly luciferase expression vector was diluted in OptiMEM medium to 1 µg/µL, and the pGL4.75[hRluc/CMV] Renilla luciferase expression vector was diluted to 100 ng/µL. Three tubes were set up as follows and mixed gently: 400 µL of Opti-MEM medium + 2 µL (2 µg) of pGL4.13[luc2/SV40] firefly luciferase expression vector + 2 µL (2 ng) of pGL4.75[hRluc/CMV] Renilla luciferase expression vector. To each tube, 6 µL of ViaFect reagent was added and the contents were gently mixed. Tubes were incubated for 10 minutes at room temperature to allow transfection complexes to develop. 200 µL of complex was added dropwise to each well of the 6-well plate, with gentle swirling to mix. Cells were returned to the incubator for 48 hours prior to processing and assay

Preparation of Cell Lysates

Transfected cells in the 6-well plate were trypsinized, divided into ten aliquots per well, pelleted at 1500 rpm for 5 minutes, and washed once with PBS. PBS was removed, and cell pellets were stored at -80°C until the time of assay.

In preparation for assay, Passive Lysis Buffer and cell pellets were warmed to room temperature, and each cell pellet was lysed in 150 µL of Passive Lysis Buffer. Cell lysis was allowed to proceed at room temperature for 15 minutes. Lysate was then serially diluted 1:2 in Passive Lysis Buffer to perform a standard curve spanning 4 to 8700 cells per well. 20 µL of each concentration of cell lysate was pipetted into triplicate wells of a 96-well plate, and 10 µL of each concentration of cell lysate was pipetted into quadruplicate wells of a 384-well plate.

Luciferase assay setup

All kit components were thawed to room temperature. Firefly Substrate was reconstituted by adding 220 µL of water to one vial containing 2.2 mg of lyophilized substrate. Aquaphile™ Coelenterazine was reconstituted by adding 220 µL of water to one vial containing 440 µg of lyophilized substrate.

Table 1. FlexStation 3 microplate reader compound addition and assay parameters

***Figure 2. SoftMax Pro 7 Software user interface for FlexStation 3 microplate reader.*The software’s straightforward interface simplifies tip and compound column assignment.

***Figure 3. DuoLuc Kinetic Trace.*The luminescent signal from the DuoLuc reporter assay was recorded over time to create a kinetic trace. Shown above are three different concentrations of cells assayed in a 384-well microplate format. Area under the curve for the firefly (35-70 seconds) and Renilla (80-120 seconds) regions of the kinetic trace are shown above. Reagent transfers occurred at 20 seconds and 70 seconds.

***Figure 4. DuoLuc reporter assay in a 96-well format.*The DuoLuc reporter assay was run in a 96-well plate using the FlexStation 3 microplate reader. Firefly (red) and Renilla (green) standard curves were plotted using a log-log curve fit in SoftMax Pro Software (r²> 0.998 for each). Three replicates were run for each dilution.

***Figure 5. DuoLuc reporter assay ran in a 384-well format.*The SpectraMax DuoLuc reporterassay was run in a 384-well plate. Firefly (red) and Renilla (green) standard curves were plotted using a loglog curve fit in SoftMax Pro Software (r2 > 0.998 for each). Four replicates were run for each dilution.

Firefly working solution was prepared by diluting firefly substrate 1:50 in Firefly Assay Buffer. Renilla working solution was prepared by diluting Aquaphile coelenterazine 1:50 in Renilla Assay Buffer. For one 96-well plate, 11 mL of each working solution was made by adding 220 µL of its respective substrate.

A preconfigured protocol in SoftMax® Pro Software was used with the parameters shown in Table 1. The software’s graphical interface facilitated the setup of compound addition (Figure 2). The FlexStation 3 microplate reader is able to pipet reagent to an entire column of the plate and read these wells repeatedly for a defined amount of time using the Flex read type. A real-time kinetic trace is generated for each sample. Within each kinetic trace, the firefly and Renilla signals could be distinguished, and the area under the curve calculated for each (Figure 3).

Results

Firefly and Renilla luciferases were measured in transfected HeLa cells using the SpectraMax DuoLucReporter assay and FlexStation 3 microplate reader. The assay run in the 96-well format demonstrated great linearity and sensitivity. We were able to detect both firefly and Renilla luminescence from 8700 cells per well down to approximately 4 cells per well (Figure 4).

Assay performance was comparable for the 384-well format, with the same degree of linearity and detection down to approximately 8 cells per well (Figure 5).

Conclusion

The SpectraMax DuoLucreporter assay, when combined with the FlexStation 3 microplate reader’s built-in fluidics and luminescence detection, is a sensitive flash-type luminescent assay for accurately measuring gene expression in mammalian cells. The reader also provides higher assay throughput than dual injector-based systems due to its ability to dispense reagent to an entire column of the plate and read the wells of the column repeatedly for a selected total experimental time. SoftMax Pro Software’s intuitive interface and preconfigured protocol allows users to quickly set up, measure, and analyze dual luciferase results.

Hoang Ha| Applications Scientist | Molecular Devices

介绍

报告基因是研究细胞通路激活相关基因表 达的重要工具。细胞转染含有报告基因和 感兴趣基因序列的质粒，通常是启动子或 其他转录控制元件。当启动子激活时，报 告基因表达且可测定其表达水平。

萤火虫荧光素酶是普遍使用的报告基因。 其发光信号所提供的灵敏度相比荧光和其 他方法更胜一筹。第二种发光报告基因是 海肾荧光素酶，通常作为组成型启动子的 对照，用以标准化诸如转染效率和细胞数 量等差异。由于萤火虫荧光素酶和海肾荧 光素酶使用不同的底物，所以两者可在同 一个孔中使用双荧光素酶实验进行检测。

SpectraMax® DuoLuc Reporter Assay 试 剂盒可实现在微孔板中使用萤火虫和海肾 荧光素酶进行高灵敏度定量实验。添加萤 火虫工作液到样品孔可启动萤火虫发光反 应，然后在加入海肾工作液可淬灭萤火虫 荧光素酶反应同时启动海肾荧光素酶发光 反应。

FlexStation® 3 多功能微孔读板机可在 96 或 384 孔板中进行高灵敏和高通量的 SpectraMax DuoLuc 实验检测。在 FlexStation 3 读板机中，整个反应系列都 是实时监测的，并同时进行机载试剂的吸 液加液和发光检测 ( 图 1 )。

材料

• FlexStation 3 多功能微孔读板机 (Molecular Devices cat. #Flex3)
  • FlexStation 3 8-通道加样头 (Molecular Devices cat. #0200-6182)
  • FlexStation 3 16-通道加样头 (Molecular Devices cat. #0200-6183)
  • 96-孔黑色 FlexStation 吸头 (Molecular Devices cat. #0900-0911)
  • 384-孔黑色 FlexStation 吸头 (Molecular Devices cat. #9000-0764)
• SpectraMax DuoLuc Reporter 检测试 剂盒 (Molecular Devices cat. #R8361)
• HeLa 细胞 (ATCC cat. #CCL-2)
• pGL4.13 [luc2/SV40] 萤火虫荧光素酶 表达载体 (Promega cat. #E668A)
• pGL4.75 [hRluc/CMV] 海肾荧光素酶表 达载体 (Promega cat. #E693A)
• ViaFect™ 转染试剂 (Promega cat. #E4981)
• Opti-MEM 低血清培养基 (ThermoFisher Scientific cat. #31985062)

***图 1 FlexStation 3 读板机上实时监测试剂添加后的反应。*通过软件计算反应 ( 萤火虫和海肾 ) 的每 个阶段特定部分曲线下面积 (AUC)

方法

细胞转染

HeLa 细胞以每孔 2x105 个细胞接种于组 培处理过的 6 孔板中，转染前在 37°C/5% 条件下孵育 24 小时。pGL4.13[luc2/SV40] 萤火虫荧光素酶表达载体经 Opti-MEM 培 养基稀释至 1 µg/µL，pGL4.75 [hRluc/CMV] 海肾荧光素酶表达载体稀释至 100 ng/µL。 三个试管按以下方式建立并轻轻混匀： 400 µL Opti-MEM 培养基 + 2 µL (2 µg) pGL4.13 [luc2/SV40] 萤火虫荧光素酶表 达载体 + 2 µL (2 ng) pGL4.75 [hRluc/CMV] 海肾荧光素酶表达载体。每管加入 6 µL ViaFect 试剂轻轻混匀。试管在室温下孵 育 10 分钟以形成转染复合物。200 µL 复 合物逐滴加入 6 孔板的每个孔中，轻轻旋 转混匀。在做下一步实验前，细胞重新孵 育 48 小时。

细胞裂解物的准备

6 孔板中的转染细胞通过胰酶消化下来，每 孔分成 10 个等分试样，1500 rpm 离心 5 分钟，PBS 洗涤一次。吸去 PBS，细胞团保 存于 -80°C 下直到开始实验。

在实验准备中，被动裂解缓冲液和细胞团 孵育至室温，将每个细胞团于 150 µL 被动 裂解缓冲液中裂解。细胞裂解过程在室温 下进行 15 分钟。裂解物在被动裂解缓冲液 中以 1:2 系列稀释，每孔生成一条从 4 至 8700 个细胞的标准曲线。20 µL 各浓度细 胞裂解物以三个复孔加入 96 孔板中，10 µL 各浓度细胞裂解剂添加到孔板的一整列孔 中，利用 Flex 读数模式按照定义好的时间 对这些孔反复读数。每个样品都会生成实 时动力学轨迹。萤火虫和海肾荧光素酶信 号在每条动力学轨迹中均可区分，且曲线 下的面积也可分别计算 ( 图 3 )

结果

HeLa 细胞中转染的 SpectraMax DuoLuc Reporter 实验的萤火虫和海肾荧光素酶在 FlexStation 3 微孔读板机上检测。在 96 孔板中进行的实验表现出非常好的线性和 灵敏度。从每孔 8700 个细胞一直到每孔大 约 4 个细胞，我们能够分别检测到萤火虫和 海肾荧光素酶发光 ( 图 4 )。 384 孔板的实验表现可与 96 孔板相媲美， 具有相同程度的线性并检测到每孔大约 8 个 细胞 ( 图 5 )。

表 1 FlexStation 3 微孔读板机化合物添加和测定参数。

***图 2 FlexStation 3 微孔读板机的 SoftMax Pro 7 软件用户界面。*软件直观的操作界面简化了吸头 和化合物列的分配

***图 3 DuoLuc 动力学轨迹。*DuoLuc reporter 实验的发光信号随时间记录以生成 动力学轨迹。上图为 384 孔板中三种不同浓度细胞的测定。萤火虫 ( 35-70 秒 ) 和 海肾 ( 80-120 秒 ) 动力学轨迹区域的曲线下面积。分别在 20 秒和 70 秒时添加试 剂。

***图 4 96 孔板中的 DuoLuc reporter 实验测定。*使用 FlexStation 3 微孔读板机 在 96 孔板中进行 DuoLuc reporter 实验测定。用 SoftMax Pro 软件中的 log-log 曲线拟合绘制萤火虫 ( 红色 ) 和海肾 ( 绿色 ) 标准曲线 ( 每条曲线 r2 >0.998 )。每个 稀释浓度做三个重复。

***图 5 384 孔板中进行 DuoLuc reporter 实验测定。*SpectraMax DuoLuc reporter 实验测定在 384 孔板中进行。用 SoftMax Pro 软件中的 log-log 曲线拟 合绘制萤火虫 ( 红色 ) 和海肾 ( 绿色 ) 标准曲线 ( 每条曲线 r2 >0.998 )。每个稀释浓 度做四个重复。

总结

当 SpectraMax DuoLuc reporter 实验与 FlexStation 3 微孔读板机内置的移液系统 和发光检测结合起来的时候，即实现了在 哺乳动物细胞中精确检测基因表达的高灵 敏闪光型发光实验。得益于其整列添加试 剂并在全部实验时间内对整列孔重复读数 的能力，FlexStation 3 读板机提供了比双 注射器系统更高的检测通量。SoftMax Pro 软件直观的操作界面和预设实验模板使 用户能够快速创建参数、检测和分析双荧 光素酶结果。

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