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Introduction

The use of gene reporters such as luciferase permits highly sensitive and nondestructive monitoring of gene expression. Firefly luciferase, a 61 kD monomeric protein, is especially attractive to many researchers because of its high sensitivity, wide linear detection range and extremely low background due to the absence of endogenous luminescent activity in mammalian cells. For the increasingly popular assay using luminescence microplate readers, the high-throughput compatible “glow” assay is often preferred for batch processing of multiple plates. In this application note, we demonstrate the measurement of luciferase expression in CHO-K1 cells using the SpectraMax^®Glo Steady-Luc™ Reporter Assay Kit, which affords longlasting luminescence signals. This assay kit is optimized for the SpectraMax^®i3x Multimode Microplate Reader with a preconfigured protocol in SoftMax^®Pro Software for rapid data analysis.

Materials

• SpectraMax Glo Steady-Luc Reporter Assay Kit, Molecular Devices (Explorer kit #R8352 or Bulk kit #R8353)
• Competitor kit: Steady-Glo Luciferase Assay System, Promega (Cat# E2510)
• SpectraMax i3x Microplate Reader, Molecular Devices (Cat# i3X)
• 96-well white walled clear bottom plates, Costar (Cat# 3903)
• Purified luciferase, Promega (Cat# E1701)
• 384-well solid white plates, Greiner (Cat# 655075)
• CHO-K1 cells, ATCC (Cat# CCL-61)
• Complete Growth Media
  • Ham’s F12 Medium, Life Technologies (Cat# 11765-54)
  • Fetal Bovine Serum, Gemini (Cat# 100-106)
  • Penicillin/Streptomycin, Life Technologies (Cat# 15070-063)
• 0.05% Trypsin EDTA, Life Technologies (Cat# 25300-054)
• pGL4 Firefly Luciferase Vector, Promega (Cat# E6681)
• pGL3 Control Vector, Promega (Cat# 1741)
• FuGeneHD Transfection Reagent, Promega (Cat# E2311)

Methods

The SpectraMax Glo Steady-Luc Reporter assay has a simplified workflow. The working solution is mixed at 1:1 ratio with the medium in each well of the microplate, where cells expressing luciferase are plated. The plate is then covered and mixed to allow complete cell lysis before the luminescent signals are read on the SpectraMax i3x Multimode Microplate Reader (Figure 1). The data acquisition and analysis is easily streamlined when using the preconfigured protocol in Softmax Pro Sofware.

Figure 1. SpectraMax Glo Steady-Luc Reporter Assay Kit workflow.

Luciferase standard curve

To identify the linear detection range of this assay, a standard curve was constructed to measure the RLU as a function of luciferase concentration. All components of the SpectraMax Glo Steady-Luc Reporter Assay Kit were first allowed to equilibrate to room temperature. SpectraMax Glo Steady-Luc working solution was made by adding D-Luciferin to the Steady-Luc Assay buffer in a 1 mg to 4 mL ratio. A ten-fold serial dilution of purified luciferase in PBS with 0.01% BSA starting at a concentration of 1 x 10⁶fg/well was prepared, and 25 μL of each concentration was added in triplicate to a 384-well solid white plate. Afterwards, 25 μL SpectraMax Glo Steady-Luc working solution was added to the wells containing luciferase and controls. The plate was shaken and incubated in the dark for 10 minutes. Using the SpectraMax i3x reader, the preconfigured SpectraMax Glo Steady-Luc Reporter assay protocol in SoftMax Pro Software was used to measure luminescence. After detection, the protocol automatically generates a curve of the data.

Transfection and cell-dilution assay

Part of assay development for a reporter gene assay is to optimize the cell number and amount of plasmid necessary to give a robust signal for screening. A cell dilution assay was performed with two different amounts of pGL4.13 firefly luciferase starting in 6-well plates. CHO-K1 cells were transiently transfected with 1.7 μg or 0.8 μg per well of pGL4.13[luc2/SV40] vector, which encodes the luciferase gene luc2 under control of the SV40 early enhancer/promoter, or pGL3-Basic (control) vector, which lacked promoter and enhancer sequences. After 24 hours, cells were trypsinized and seeded at 100 μL/well into a 96-well plate starting at 30,000 cells/well with subsequent 2-fold dilutions. 100 μL/well Steady-Luc working solution was added to the wells. The plate was covered to protect the reagents from light and mixed using an orbital shaker. After 10 minutes, luminescence was measured and recorded on the SpectraMax i3x reader using the preconfigured SpectraMax Glo Steady-Luc Reporter assay protocol. The preconfigured protocol is found in the Reporter Assays section under the Protocol Library Tab or on the SoftMax Pro Software protocol sharing website (www.softmaxpro.org).

Screening applications

A reporter gene assay screen was simulated by performing multiple reads of a plate of pGl4 firefly luciferase transfected cells using the SpectraMax Glo Steady-Luc Reporter Assay Kit. Cells were transfected as previously described and plated at 30,000 cells per well in a 96-well plate and grown overnight. On the day of the assay, the plate was equilibrated to room temperate, and then 100 μL reconstituted Steady-Luc working solution containing D- Luciferin was added to each well. The plate was covered and mixed on an orbital shaker for five minutes and then placed In a SpectraMax i3x reader and mixed. Luminescence was read every five minutes for a total of five hours with three seconds of orbital shaking before each read.

Results

Luciferase standard curve

For comparison, the standard curve to identify the detection range of the assay was also performed with a competitor’s glow luciferase assay. In both cases, the assays identified a linear relationship between luminescence and luciferase concentration (Figure 2). The R₂values for luciferase were 0.998 using the SpectraMax Glo Steady-Luc Reporter Assay Kit and 0.999 using the competitor assay kit. A paired t-test of normalized data indicates equivalent results at each concentration (P < 0.05). Calculations were performed using GraphPad Prism. Using both kits, the Lower Limit of Detection (LLD) of luciferase was 5 femtograms/well in 384-well format.

***Figure 2. Luciferase standard curve in 384-well format.*Linear results from a 10-fold dilution of purified luciferase in either the SpectraMax Glo Steady-Luc Reporter assay (Green) (R₂=0.999) or a competitor assay (Red) (R2=0.998). In both assays the LLD=5 femtogram/well.

Measurement of luciferase in transfected cells

Titration of CHO-K1 cells transiently transfected with luciferase starting at 30,000 cells/well was pipetted into the wells followed by Steady-Luc Reporter working solution. Shown in Figure 3, both the SpectraMax Glo Steady-Luc Reporter assay and competitor glow luciferase assay demonstrated a linear relationship between cell number and luminescence. For cells that received 0.8 μg of pGL4.13 luciferase vector, R2=0.995 using the SpectraMax Glo Steady-Luc Reporter Assay Kit and R2> 0.999 using the competitor assay kit. Both kits detected the lowest dilution of cells tested in the assay, 468 cells/well.

***Figure 3. Measurement of luciferase in transfected cells.**90% confluent CHO-K1 cells were transfected with either 1.7 μg pGL4 (Firefly) (Red), 0.8 pGL4 (Firefly) (Green), or pGL3 (control) luciferase plasmids (Blue). **(A)**SpectraMax Glo Steady-Luc Reporter assay (R2>0.995 at 0.8 μg plasmid) and **(B)*Competitor assay (R₂=0.999 at 0.8 μg plasmid). Both kits detected the lowest number of cells/well in the assay (468 cells/well).

Screening applications

The SpectraMax Glo Steady-Luc Reporter assay is a glow-based luminescence assay that provides an extended signal time window (Figure 4). A plate with luciferase expressing CHO-K1 cells at 30,000 cell/well is read every five minutes to simulate batch processing of screening plates. At five hours, the signal is within 20% of the initial value. On each individual plate in a reporter assay screen, a control for normalization of data to background is included so that data can be compared across many plates. As demonstrated in Figure 5, the signal stays within 15% across 20 plates, suggesting the feasibility of running 20 plates in a 90 minute period with this assay.

***Figure 4. Luciferase signal in CHO-K1 cells over time.*CHO-K1 cells were transfected with luciferase. On the day of the assay, the plate was equilibrated to room temperature, and then 100 μL reconstituted Steady-Luc working solution containing D-Luciferin was added to each well. The plate was covered and mixed on an orbital shaker for five minutes and then placed in a SpectraMax i3x reader and mixed. Luminescence was read every five minutes for five hours with three seconds of orbital shaking before each read.

***Figure 5. Screening applications.*Raw data from luciferase transfected CHO-K1 cells in the SpectraMax Glo Steady-Luc assay. The average signal from the luciferase transfected cells is plotted as an example of batch processing during a screening assay in which a plate is read every five minutes. On screening plates, background controls are used to normalize data so that comparisons can be compared across many plates.

Conclusion

Luciferase-based reporter assays using luminescence microplate readers have become increasingly popular for high-throughput analysis of chemical biology and drug discovery applications. The SpectraMax Glo Steady-Luc Reporter assay kit allows for sensitive quantification of firefly luciferase expression in mammalian cells. By applying a homogeneous experimental protocol, the specially formulated mixture of substances in this kit significantly extends the time window with steady signal, thereby enabling batch processing of plates in screening assays. This assay kit is optimized for SpectraMax microplate readers from Molecular Devices, with a pre-configured protocol provided for customers using Molecular Devices’ SoftMax Pro Software for their data acquisition and analysis.

Learn more about SpectraMax Glo Steady-Luc Reporter Assay Kit >>

简介

使用荧光素酶等报告基因等是高灵敏度且 无损检测基因的表达一种手段。萤火虫荧 光素酶是分子量为 61 kD 的单分子蛋白， 由于其高灵敏度，线性检测范围广，背景 低，在哺乳动物细胞中缺乏内源性发光活 性，因此对许多研究人员特别具有吸引 力。由于使用读板机检测荧光的方法越来 越流行，因此高通量兼容的“辉光”检测 方法常常是多板批量处理的首选方法。在 本应用说明中，我们展示了使用 SpectraMax®Glo Steady-Luc™ 报告基因试剂盒检 测 CHO-K1 细胞中荧光素酶的表达，该试 剂盒提供了持久的发光信号。本实验试剂 盒是针对 SpectraMax®i3x 多功能读板机 进行优化的，并采用 SoftMax®Pro 软件中 的配置的模板，用于快速数据分析。

材料与方法

• SpectraMax Glo Steady-Luc 报告基因 试剂盒, Molecular Devices (Explorer kit #R8352 or Bulk kit #R8353)
• 对比试剂盒: Steady-Glo Luciferase Assay System, Promega (Cat# E2510)
• SpectraMax i3x 微孔板读板机, Molecular Devices (Cat# i3X)
• 96 孔白色底透板, Costar (Cat# 3903)
• 纯化荧光素酶, Promega (Cat# E1701)
• 384 孔白色微孔板, Greiner (Cat# 655075)
• CHO-K1 细胞, ATCC (Cat# CCL-61)
• 完全培养基
  • Ham’s F12 培养基, Life Technologies (Cat# 11765-54)
  • 牛血清, Gemini (Cat# 100-106)
  • 盘尼西林 / 链霉素, Life Technologies (Cat# 15070-063)
• 0.05% 胰蛋白酶 EDTA, Life Technologies (Cat# 25300-054)
• pGL4 萤火虫荧光素酶载体, Promega (Cat# E6681)
• pGL3 空白载体, Promega (Cat# 1741)
• FuGeneHD 转染试剂, Promega (Cat# E2311)

方法

SpectraMax Glo Steady-Luc Reporter 实 验有一个简化的工作流程。微孔板的每一 个孔内都放入表达荧光素酶的细胞并加入 工作液与培养基 1:1 混合后的溶液。在使 用 SpectraMax i3x 多功能读板机读取发光 信号前，避光并混匀使细胞完全裂解。使 用 Softmax Pro 软件中内置的模板，数据 采集和分析很容易简化。

图 1 SpectraMax Glo Steady-Luc Reporter 试剂盒工作流程

荧光素酶标准曲线

为了确定该方法的线性检测范围，我们 构建了一条标准曲线来测量荧光素酶浓度 与 RLU 的关系。首先将 SpectraMax Glo Steady-Luc Reporter 试剂盒的所有试剂 放置在室温下平衡。将 D-Luciferin 以 1 mg - 4 mL 的比例添加到 Steady-Luc 缓冲 液中，制得 SpectraMax Glo Steady-Luc 工作液。将 PBS 溶解的纯化荧光素酶以 1 x 106 fg/well 起始浓度做 10 倍梯度稀释， 每个浓度的溶液取 25 μL 放入 384 孔白色 微孔板，做 3 个复孔。25 μL 对照品与 SpectraMax Glo Steady-Luc 工作液都加 入到微孔板后，震荡摇匀并避光放置 10 min。使用 SpectraMax Glo Steady-Luc Reporter 内置的 SpectraMax Glo SteadyLuc Reporter 实验模板设定程序并通过 SpectraMax i3x 读板机检测发光。检测结 束后，模板会根据数据自动生成一条曲 线。

转染及细胞稀释实验

部分报告基因实验的方法开发实验是为了 优化细胞数目和质粒数量，从而为筛选提 供可靠的信号。细胞稀释实验是在 6 孔板 上使用两种不同数量的 pGL4.13 萤火虫荧 光素酶进行。每个孔使用 1.7 µg ，0.8 µg pGL4.13 [luc2/SV40] 载体以及 pGL3 空白 载体转染 CHO-K1 细胞，其中 pGL4.13 [luc2/SV40] 载体编码了荧光素酶基因 luc2 并在前面连接了 SV40 早期增强子 / 启动 子，而 pGL3 空白载体缺少启动子和增强子 序列。24 小时以后，使用胰蛋白酶将细胞 消化并以 30000 细胞 / 孔的起始数量 2 倍 稀释，在 96 孔板内放入每孔 100 μL。每 孔再加入 100 μL 的 Steady-Luc 工作液。整 板避光并使用圆周型振荡器充分混匀。10 min 过后，使用 SpectraMax i3x 读板机内 置的 SpectraMax Glo Steady-Luc Reporter 实验模板检测发光。该内置的模 板可在 Protocol Library 标签下的 Reporter Assay 中，或是在 SoftMax Pro 软件模 板分享网站中找到 (www.softmaxpro. org)。

筛选类应用

使用 SpectraMax Glo Steady-Luc Repoter 试剂盒对转染 pG l4 萤火虫荧光素酶的细 胞平板进行多次读取，模拟报告基因检测 筛选。细胞如前所述转染，在 96 孔板中以 每孔 30,000 个细胞的密度铺板，并过夜生 长。在实验当天将细胞板放置在室温平 衡，然后每个孔加入 100 µL 重组的含有 DLuciferin 的 Steady-Luc 工作液。将细胞 板避光并放置在圆周型振荡器上混匀 5 mins，然后放置在 SpectraMax i3x 读板 机内混匀。每 5 分钟读取一次发光信号， 每次读取前进行 3 秒的圆周式震动，总共 持续 5 个小时。

结果

荧光素酶标准曲线

为了进行比较，我们还使用了竞争对手的 glow luciferase 实验来确定标准曲线的检 测范围。在这两种情况下，荧光素酶的测 定结果都与荧光素酶浓度呈线性关系 ( 图 2 )。使用 SpectraMax Glo Steady-Luc Reporter 试剂盒测定荧光素酶 R2 值为 0.998，使用竞争对手试剂盒测定荧光素 酶 R2 值为 0.999。归一化数据的双侧 t 检 验表明各浓度下结果相同 (P < 0.05)。计算 结果是通过 GraphPad Prism 软件获得。 两种试剂盒在 384 孔板上对荧光素酶的最 低检测限 (LLD) 均为 5 fg / 孔。

***图 2 荧光素酶在 384 孔板内的标准曲线。*直线是利用 SpectraMax Glo Steady-Luc Reporter 试剂 盒 ( 绿色 ) (R2=0.999) 和相似试剂盒 ( 红色 ) (R2=0.998) 检测 10 倍梯度稀释纯化荧光素酶的数据绘 制而成。二者的最低检测限均为5 fg / 孔。

转染细胞的荧光素酶测定

将荧光素酶瞬时转染 CHO-K1 细胞，从 30,000 个细胞 / 孔开始，用枪滴入孔中，然 后添加 Steady-Luc Reporter 工作液。如 图 3 所示，SpectraMax Glo Steady-Luc Reporter 实验和竞争对手 glow luciferase 实 验都显示了细胞数量与发光之间的线性关 系。在 0.8 µg pGL4.13 转染的细胞中,使用 SpectraMax Glo Steady-Luc Reporter 试 剂盒检测的荧光素酶 R2 值为 0.995，使用 竞争对手试剂盒的 R2 值大于 0.995。两种 试剂盒在实验中检测的最低细胞浓度为 468 细胞 / 孔。

***图 3 转染细胞的荧光素酶测定。*铺板的 90% 的 CHO-K1 细胞被 1.7 µg pGL4 ( 萤火虫 ) ( 红色 )， 0.8 µg pGL4 ( 萤火虫 ) ( 绿色 ) 或 pGL3 ( 对照 ) 荧光素酶质粒 ( 蓝色 ) 转染。(A) SpectraMax Glo Steady-Luc Reporter 实验 ( R2 >0.995 在 0.8 µg 质粒 ) 和 ( B ) 类似实验 ( R2=0.999 在 0.8 µg 质粒 )。 两种试剂盒在实验中检测的最小的细胞数目是 468 细胞 / 孔。

筛选类应用

The SpectraMax Glo Steady-Luc Reporter 实验是一种基于荧光的发光实验，提供了 较长的时间窗口 ( 图 4 )。一块含有表达荧 光素酶的 CHO-K1 细胞 ( 30,000 细胞 / 孔 ) 的微孔板每 5 mins 读数一次以模拟批量操 作的筛选类实验。5 小时时，信号在初始值 的 20% 以内。在报告基因筛选的实验中每 一块板上都包含一个对照作为背景值，以 便多个板之间比较数据。如图 5 所示，20 块板的信号值变化在 15% 以内，这说明在 90 min 内读取 20 块板的可行性。

***图 4 CHO-K1 细胞内荧光素酶信号随时间的动态变化。CHO-K1 被转染表达荧光素酶。*在实验当 天，微孔板放置在室温平衡，然后在每个孔加入 100 µL 重构建的包含 D-Luciferin 的 Steady-Luc 工作液。微孔板在圆周震荡器上避光混匀 5 min，然后放入 SpectraMax i3x 读板机内混匀。发光信 号在 5 小时内每 5min 读数 1 次，并在每次读数前震荡。

***图 5 筛选类应用。*转染荧光素酶的 CHO-K1 细胞在 SpectraMax Glo Steady-Lu 中的原始数据。荧 光素酶转染细胞每 5 mins 读取的平均信号被绘制，当作一个筛选实验里批次处理的微孔板。在筛 选板上，使用背景对照对数据做归一化处理，从而多块板之间可以进行比较。

结论

使用化学发光微孔板读板机完成基于荧光 素的报告基因实验在高通量生物化学以及 药物筛选中越来越受欢迎。SpectraMax Glo Steady-Luc Reporter 实验试剂盒可以 灵敏的定量检测到哺乳动物细胞中萤火虫 荧光素酶的表达。通过采用均质的实验方 案，本试剂盒中特殊配制的混合物质显著 延长了时间窗，且信号稳定，从而实现了 筛选类实验中微孔板的批量处理。该检测 试剂盒是针对美谷分子仪器的微孔板读板 机优化设计，在美谷分子的 SoftMax Pro 软件上已预置一个模板提供给用户，方便 数据的获取及分析。

Learn more about SpectraMax Glo Steady-Luc Reporter Assay Kit >>

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