Detect dual luciferase expression on the FlexStation 3 microplate reader

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.