By Michael Su, Jinfang Liao, M.D., Ph.D. and Evelyn McGown, Ph.D., Molecular Device, Inc, 1311 Orleans Dr., Sunnyvale, CA 94089
Introduction
Reporter-gene assays are used to study eukaryotic gene expression. Dual genetic reporters are commonly used in transient transfections of cultured cells to minimize experimental variability caused by differences in cell number, viability or transfection efficiency. One plasmid containing the experimental reporter gene (coupled to a regulated promoter) is cotransfected with a second plasmid containing a control reporter gene (coupled to a constitutive promoter). Bioluminescent reporter systems using firefly and Renilla luciferases are widely used as co-reporters because both assays are easy and sensitive. Previously, luciferase reporter assays have been “flash” assays that must be read within seconds of reagent addition and require integrated injectors in the luminometer. (See Maxline App Note #39). Recently, Promega introduced a Dual-Glo Luciferase assay system for high-throughput analyses. The signals are stable for 2 hours after reagent addition and integrated injectors are not necessary.
Figure 1 illustrates the two-enzymatic reactions. Firefly luciferase enzyme catalyzes the oxidation of luciferin with the concomitant release of light.1 The reaction requires ATP, Mg2+ and O2. Renilla luciferase catalyzes the O2-dependent oxidation of coelenterate luciferin (coelenterazine) but does not require Mg2+ or ATP. 2 The enzymes have different substrate requirements, so they can both be measured in a single reaction mixture. Both reactions can easily be measured in Molecular Devices microplate luminometers. Examples of results are presented below for the LMax™ II384 dedicated, top-of-the-line microplate luminometer, the Analyst® GT Multi-Mode, Filter-Based Instrument, and the SpectraMax® M5 Multi-Detection, Monochromator-Based Reader with both top- and bottom-read capability.
Materials
Dual-Glo luciferase assay system (Promega Cat. #E2920)
Quantilum Recombinant firefly luciferase (Promega Cat. #E1720)
Renilla luciferase (Chemicon Cat. #4400)
Storage Buffer, 25 mM Tris-acetate, 1 mM EDTA, 1 mM DTT, 0.2 M NH4SO4, 15% glycerol, 30% ethylene glycol, 0.1% BSA
Standard Buffer, Phosphate-buffered saline, pH 7.4 (PBS) plus 0.01% BSA
Cell line: CHO-K1 (ATCC Cat. #CCL-61)
Firefly luciferase plasmids: pGL3-control vector (Promega Cat. #E1741) and PCRELuc (BD Biosciences Cat. #631911)
Renilla luciferase plasmid: phRL-TK vector (Promega Cat. #E6241)
Lipofectamine (Invitrogen Cat. #10378-016)
Opti-Mem (Invitrogen Cat. #31985-070)
SpectraMax M5 Multi-Detection Microplate Reader, Analyst GT Multimode Microplate Reader and LMax II384 Luminescence Microplate Reader (Molecular Devices)
Methods
Stock standards preparation
Step 1. Stock firefly luciferase standard (1.0 mg/ mL) was prepared by adding 0.3 mL of a 14.9 mg/mL stock of firefly luciferase to 4.2 mL of the standard buffer in a plastic vial. The 1.0 mg/mL standard was then divided into 0.5 mL aliquots and stored at -70°C until needed.
Step 2. Daily working firefly luciferase (10 μg/ mL) was prepared by transferring 10 μL of the 1.0 mg/mL firefly luciferase to 990 μL standard buffer.
Step 3. Renilla luciferase (10 μg /mL) was prepared by adding 990 μL of the standard buffer to the Renilla luciferase bottle containing 10 μg of Renilla luciferase
Preparation of combined firefly and Renilla luciferase standards
Step 1. A combined luciferase stock standard (100 ng/mL each) was prepared by transferring 10 μL of each luciferase standard to 980 μL of standard buffer in a plastic vial.
Step 2. The working combined standard curve was prepared by making serial 10-fold dilutions in the standard buffer to achieve 10 ng/mL down to 10 fg/mL each.
Preparation of reagents
Dual-Glo reagent and Dual-Glo Stop & Glo reagent were prepared as directed in the kit instructions.
Assay procedure with luciferase standards
The assay plate was prepared by pipetting 25 μL/ well combined firefly/Renilla luciferase standards and blanks into a Costar 384-well white plate in replicates of six, followed by 25 μL/well of DualGlo luciferase substrate solution to each well. The plate was mixed briefly with a plate shaker, incubated 10 minutes at room temperature in the dark and then read for firefly luciferase activity. According to the kit insert, the firefly luciferase luminescence signal is relatively stable for two hours with greater than 60% of the initial luminescence signal retained in that time.3 After the firefly luciferase was measured, DualGlo Stop & Glo substrate solution (25 μL/ well) was added to each well to quench the firefly luciferase reaction and provide substrate for the Renilla luciferase reaction. The plate was agitated briefly, incubated 10 minutes at room temperature in the dark and read again. The Renilla luciferase luminescence signal is also relatively stable for two hours.3
Cell-based assays
CHO-K1 cells were seeded at a density of 2 x 106/100 mm plate overnight, and transientlytransfected with pGL3-control vector and phRL-TK vector (6 μg DNA total; pGL3 to phRL-TK ratio = 50:1) following the standard Lipofectamine transfection protocols. The pGL3- control vector contains cDNA-encoding firefly luciferase with expression driven by the SV40 promoter. After 24 hours, the cells were seeded overnight in 25 μL at densities of 0 to 25,000 cells/well in a Costar white wall/clear bottom 384-well plate (n=6/group). The following day, Dual-Glo luciferase substrate solution was added (25 μL/well). The plate was agitated briefly and incubated 10 minutes at room temperature in the dark to ensure complete cell lysis and enzyme equilibration. It was then read for firefly luciferase activity. After the firefly luciferase was measured, Dual-Glo Stop & Glo substrate solution (25 μL/well) was added to each well to quench the firefly luciferase reaction and provide substrate for the Renilla luciferase reaction. The plate was agitated briefly, incubated 10 minutes at room temperature in the dark and read again.
To measure induction of pCRE-luc (cAMP response element) by Forskolin and 8-Br-cAMP, CHO-K1 cells were seeded overnight at a density of 2 x 106/100 mm plate and transientlytransfected with pCRE-luc and phRL-TK vectors (6 μg DNA total; pCRE-luc to phRLTK ratio = 50:1) following the standard Lipofectamine transfection protocols. 24 hours after transfection, cells were seeded overnight in 100 μL (60,000 cells/well) in a Costar white wall /clear bottom 96-well plate, then treated with 50 μM Forskolin or 1 mM 8-Br-cAMP for 6 hours (Control = untreated). Firefly (pCRE-Luc) and Renilla (phRL-TK) luciferase activities were measured with the Dual-Glo assay system.
Instrument settings
SpectraMax M5 Microplate Reader:
Read type = endpoint, Read Mode = luminescence with 1000 ms integration time, Wavelengths = All, Automix = off, Auto calibration = on, Settling time = off, Autoread = off.
LMax II384 Microplate Luminometer: Integration time = 1 second, Settling time = off, Autoread = off.
Analyst GT Microplate Reader: Luminescence height = 1 mm, max integration as 1,000,000 μsec., target CV per well = 1.0%, raw data units = counts/sec., attenuation mode = out.
Data analysis
Molecular Devices SoftMax® Pro software was used to analyze and present the data. The lower limit of detection (LLD) was defined as the level of luciferase detectable above 3 positive standard deviations of the blank.
Results
Luciferase emission spectrum
Figure 2 shows the emission spectrum of firefly luciferase obtained on the SpectraMax M5. The emission lambda maximum was 560 nm, as has been reported in the literature. The emission lambda max of Renilla luciferase is approximately 480 nm (data not shown).
Luciferase standard curves
Figure 3 shows representative firefly and Renilla luciferase standard curves obtained in a 384-well plate in the LMax II384. The firefly luciferase signal was an order of magnitude greater than that of the Renilla. In this particular experiment, the estimated lower limits of detection (LLDs) were 2 and 20 fg/ well for firefly and Renilla luciferase, respectively in both the LMax II384 and the Analyst GT Systems. Figure 4 shows a firefly luciferase dilution series measured in a 96-well plate in the SpectraMax M5 and the Analyst GT. Typical LLDs were approximately 1–2 fg/ well in the Analyst GT and 2–3 fg/well in the SpectraMax M5. The dynamic range of the assay was approximately 5 orders of magnitude.
Cell-based experiments
In the first experiment, we prepared a dilution series of CHO-K1 cells transiently transfected with pGL3-control vector and phRL-TK vector. The cells were seeded overnight at densities varying from 0 to 25,000 cells/well in a 384-well plate and firefly and Renilla luciferase activities were measured in an LMax II384 (Figure 5) and an Analyst GT (data not shown). The estimated LLD was 25 cells/well in firefly luciferase activity in both instruments and the Z' factors of LLD were similar (0.52 in LMax II and 0.58 in Analyst GT). SpectraMax M5 would give similar results, although these particular experiments were done before the SpectraMax M5 became available.
Note: the Dual-Glo reagent lyses the cells and releases the enzymes into the solution, so the luminescence measurement is basically the same as with purified enzymes in solution. The Spectramax M5 has similar detection capability to the Analyst GT and LMax II384 (Figure 4), so we are confident that its performance is also similar when measuring the enzymes in cell lysates.
The purpose of the next experiment was to demonstrate that the Dual-Glo assay system could be used to measure the induction of pCREluc by forskolin and 8-Br-cAMP. Figure 6, Panel A shows the firefly and Renilla luciferase activities measured with the Dual-Glo assay system in an LMax II384. The marked induction of firefly luciferase activity by forskolin and 8-Br-cAMP is obvious. As expected, there is little or no effect on Renilla activity. Similar results were obtained on the Analyst GT.
Figure 6, Panel B shows the same results, after the firefly activity has been normalized to Renilla activity (i.e., ratio of firefly activity to Renilla activity). The induction of firefly activity is approximately 3-fold and the Z' factors for both instruments were 0.64 and 0.62 for forskolin at 50 μM and 8-Br-cAMP at 1 mM respectively.
Discussion and conclusions
The results above revealed excellent detection limits and dynamic range in all three Molecular Devices microplate luminometers. The estimated lower limit of detection (LLD) in both 96- and 384-well plates is < 2 fg/well for firefly luciferase in the LMax II384 and Analyst GT and 2–3 fg/ well in the SpectraMax M5. Renilla luciferase emission is much dimmer than the firefly and thus its LLD is 10 times higher.
The LLD for firefly luciferase activity in transientlytransfected CHO-pGL3-luc-phRL-TK cells was 25 cells/well (Z' factor = 0.58 and 0.52 for LMax II384 and Analyst GT respectively). Results would be similar for the SpectraMax M5, based on results of several experiments with purified enzymes (e.g., Figure 4). As pointed out above, once the Dual-Glo reagent is added, the cells become lysed and the enzymes are free in solution during the actual luminescence measurements, just as they are with the enzyme standard preparations.
The instruments can also easily detect Forskolin and 8-Br-cAMP induced pCRE-luc activity in transiently transfected CHO-pCRE-luc-phRLTK cells (Z' factors were 0.64 and 0.62 for forskolin and 8-Br-cAMP respectively).
The LMax II384 is a dedicated, top-of-theline microplate luminometer. It provides the sensitivity, flexibility, automation capability and validation tools required by leading laboratories and is suitable for high-throughput applications. The LMax II384 with dual liquid injectors is designed for both flash and glow luminescence detection applications. Ultra-fast photon counting technology provides superior signalto-noise ratio. The LMax II384 can be integrated with robotic systems.
The Analyst GT delivers unsurpassed multimode assay detection performance including an independent optical system and photomultiplier tube for luminescence detection. The dedicated luminescence PMT has extremely low background counts and superior performance in the blue wavelengths. The Analyst GT is equipped with 20 plate magazines in a bi-directional stacker.
The SpectraMax M5 is a multi-detection, monochromator-based reader with both topand bottom-read capability. It offers wavelength scanning capability, as well as luminescence performance competitive with dedicated luminometers.
References
1. M.A. DeLuca and W.D. McElroy. (1978) in Meth Enzymol Vol. 53; p. 3.
2. J.C. Matthews, et al. (1977) Purification and properties of Renilla reniformis luciferase. Biochemistry, 16:58.
3. Promega, Dual-Glo luciferase assay system technical manual, www.promega.com.