Multiplex Hextuple Luciferase Assaying

Abstract

We recently expanded the commonly used dual luciferase assaying method towards multiplex hextuple luciferase assaying, allowing monitoring the activity of five experimental pathways against one control at the same time. In doing so, while our expanded assay utilizes a total of six orthogonal luciferases instead of two, this assay, conveniently, still utilizes the well-established reagents and principles of the widely used dual luciferase assay. Three quenchable D-Luciferin-consuming luciferases are measured after addition of D-Luciferin substrate, followed by quenching of their bioluminescence (BL) and the measurement of three coelenterazine (CTZ)-consuming luciferases after addition of CTZ substrate, all in the same vessel. Here, we provide detaileded protocols on how to perform such multiplex hextuple luciferase assaying to monitor cellular sginal processing upstream of five transcrciption factors and their corresponding transcription factor-binding motifs, using a constitutive promoter as normalization control. the first protocol is provided on how to perform cell culture in preparation towards genetic or pharmaceutical perturbations, as well as transfecting a multiplex hextuple luciferase reporter vector encoding all luciferase reporter units needed for multiplex hextuple luciferase assaying. the second protocol details on how to execute multiplex hextuple luciferase assaying using a microplate reader apppropriately equipped to detect the different BLs emitted by all six luciferases. Finally, the third protocol provides details on analyzing, plotting and interpreting the data obtained by the microplate reader.

1. Introduction

To comprehensively characterize complex biomedical cellular phenomena, or identify genetic or pharmaceutical agents that modulate those phenomena, assaying methods that provide multiple, simultaneous measurements are highly preferred. Unfortunately, most modern cell-based assays utilize only a single measurement (e.g., enzyme inhibition or cell viability) to understand the interaction between model system and perturbation. Additional assays can be performed to expand information content, but this often requires protracted assay development, time, and/or expense. Moreover, independently performed assays may or cannot always be appropriately controlled, making comparative analysis across different experimental variables difficult (1, 2). Multiplexed cellular assays seek to address these limitations by measuring multiple readouts from a single assaying unit (3, 4), providing more detailed cellular insights and allow greater perspective and nuance in assessing normal versus disease-associated processes, or identifying candidate therapeutic hits during drug screening efforts (5-7). Furthermore, correlations between experimental variables and biological effects can be more accurately obtained and compared since multiple measurements derive from the same sample (8).

Detection agents integrated in multiplex assays are required to act orthogonally and preferentially over large dynamic ranges. Luciferases are cost-effective, versatile, quantitative, and genetically encoded, and therefore great candidates for multiplexed assays. Luciferases possess many advantages over fluorescent proteins, such as substrate dependence, higher sensitivity, wider dynamic detection range (9, 10), and the absence of auto-luminescence in mammalian cells (9). Similar to fluorescent proteins, each luciferase (combined with substrate) illustrates a unique emission spectrum (11) that make possible the simultaneous detection of two or more luciferases, as long as appropriate detection filters are used to distinguish between their emission spectra (12, 13).

Until recently, the majority of the luciferase-based assays are founded on dual luciferase assaying, which improves experimental accuracy (14). Typically, firelfly luciferase, a D-Luciferin-consuming luciferase, is used to monitor a specific cellular signaling pathway, while renilla luciferase (Renilla/RLuc), a coelenterazine (CTZ)-consuming luciferase, is coupled to an internal control used for data normalization (see Fig. 1A). A quencher is added along with the second substrate, inhibiting the emission catalyzed by firefly luciferase (FLuc), allowing for reading of light unique to each luciferase in a sequential process in the same vessel. This eliminates pipetting errors that would result from measuring luciferase activities separately (15).

Fig. 1. Schematic comparison between analyzing cellular signaling processing through transcription factors using dual luciferase assaying, and multiplex hextuple luciferase assaying.

(A) Simplified schematic of five cellular signaling pathways upstream of five transcription factor-binding motifs, of which only one can be monitored, using the FLuc, against a control pathway, using the Renilla luciferase reporter (Renilla/RLuc), by dual luciferase assaying. Five separate experiments have to be performed to obtain values for all five signaling pathways. (B) Simplified schematic of five cellular signaling pathways upstream of five transcription factor-binding motifs, of which all five can be monitored, using the FLuc, the RedF, the NLuc, the Renilla luciferase reporter (Renilla/RLuc), and the green Renilla luciferase reporter (GrRenilla/GRLuc), against a control pathway, using the enhanced beetle luciferase reporter (ELuc), by multiplex hextuple luciferase assaying. Just one experiment has to be performed to obtain values for all five signaling pathways. In both scenarios, cells are transfected with (a) luciferase plasmid(s), transfected cells washed and lysed, followed by the addition of D-Luciferin substrate to record one (dual luciferase assay) (A), or three (multiplex hextuple luciferase assay) (B) signal(s), emitted by the D-Luciferin-consuming luciferase(s), followed by quenching the D-Luciferin-catalyzed emission signal(s) and the addition of coelenterazaine to record again, one (dual luciferase assay) (A), or three (multiplex hextuple luciferase assay) (B) signal(s) emitted by the CTZ-consuming luciferase(s). The obtained signals, emitted by one (dual luciferase assaying) (A) or five (multiplex hextuple luciferase assaying) (B) pathway luciferase reporter(s), are mathematically processed and plotted against the signal emitted by the control luciferase reporter.

Recently, we demonstrated that dual luciferase assaying can be expanded towards multiplex hextuple luciferase assaying providing opportunities monitoring five cellular signaling pathways against a control signaling pathway, while still using the same reagents standardly used during dual luciferase assaying (16). Three quenchable D-Luciferin-consuming luciferases, enhanced beetle luciferase (ELuc), FLuc, and red firefly luciferase (RedF), are measured during a first step. This step is followed by quenching of their bioluminescence (BL) and the measurement of three CTZ-consuming luciferases, Nano luciferase (NLuc), the Renilla luciferase (Renilla/RLuc), and the green Renilla luciferase (GrRenilla/GRLuc)), during a second step. Both steps occur in the same vessel (see Fig. 1B).

To facilitate multiplex hextuple luciferase assaying, we assembled the multiplex hextuple reporter vector using a flexible synthetic assembly cloning pipeline that can be applied to any set of reporter genes. Transfecting a single plasmid, encoding all luciferase reporters together, steps away from traditional cotransfection methods typically used for luciferase or any other reporter assays, where each luciferase reporter is encoded by one plasmid (see Fig. 2A). Hence, using this approach, which we refer to as solotransfection, we can ensure equal stoichiometric ratios of each transcriptional unit in each transfected cell, results in lower experimental variability and smaller error bars (see Fig. 2B).

Fig. 2. Schematic comparison between performing multiplex hextuple luciferase assaying by cotransfecting six individual luciferase reporter vectors, or solotransfecting one multiplex hextuple luciferase reporter vector.

Simplified schematic of multiplex hextuple luciferase assaying to monitor changes in five cellular signaling pathways acting upstream of five transcription factors (TF 1, TF 2, TF 3, TF 4, and TF 5) and their respective transcription factor-binding motifs, against a control cellular signaling pathway (constitutive CMV promoter), after cotransfecting six individual luciferase reporter plasmids (A), or solotransfecting a single multiplex luciferase reporter vector generated by synthetic assembly cloning (B) (see also accompanying chapter). Cells, transfected with (a) plasmid(s) encoding specific cellular signaling pathways and a defined control reporter pathway using cotransfection (A) or solotransfection (B), and treated with genetic and/or pharmaceutical perturbations, are washed and lysed, followed by adding D-Luciferin substrate and measuring a first set of signals emitted by the D-Luciferin-consuming luciferases, followed by quenching the previous D-Luciferin-catalyzed emissions and addition of CTZ substrate, followed by measuring a second set of signals emitted by the CTZ-consuming luciferases. All six signals are mathematically processed to obtain quantitative changes in five cellular signaling pathways upstream of specific transcription factors normalized against a control. Since equal stoichiometric cellular uptake of all luciferase reporters is only ensured during solotransfection, but not cotransfection, experimental variability (measured by the coefficient of variation, %CV) and smaller error bars are lower during solotransfection, compared to cotransfection.

Two types of vectors are needed for successful completion of multiplex hextuple luciferase assaying. The first type of vectors includes constitutively expressing luciferase reporter plasmids for all six luciferases (ELuc, FLuc, RedF, NLuc, Renilla/RLuc, and GrRenilla/GRLuc) (see Table 1 and Fig. 3A), that will be used to determine the transmission coefficients, when performing the multiplex hextupe luciferase assay for the first time, or under novel conditions compared to initially established transmission coefficients (culture medium, temperature, and so on). These transmission coefficients are essential to determine luciferase-specific emission contributions in a mixture, that then can be correlated with cellular pathway activities acting upstream of each luciferase reporter. The second type of vectors are six generic pathway transcriptional reporter plasmids (see Fig. 3B) included in a generic multiplex hextuple luciferase vector (see Table 1 and Fig. 3C) to perform multiplex pathway analysis using multiplex hextuple luciferase assaying, of which one is used as a normalization control, and the other five report on pathway signaling upstream of five transcription factors and their respective transcription factor-binding motifs. A practical example of a multiplex hextuple luciferase vector that monitors five specific pathways against a control is indicated (see Fig. 3D).

Table 1. Summary of plasmids described in this work.

For each plasmid, the following categories are indicated: plasmid type (Type), plasmid abbreviation (Abbreviation), plasmid description (Description), plasmid function (Function), vector backbone (Backbone), vector resistance (Resistance), and coordinate to obtain the plasmid from the public repository Addgene (Addgene).

Type	Abbreviation	Description	Function	Vector	Resistance	Addgene
Single luciferase reporters	hCMV-IE1:ELuc	Constitutively expressed ELuc	Transcriptional Unit	pColE1_Alpha2	Kanamycin	#118062
	hCMV-IE1:FLuc	Constitutively expressed FLuc	Transcriptional Unit	pColE1_Alpha2	Kanamycin	#118063
	hCMV-IE1:RedF	Constitutively expressed RedF	Transcriptional Unit	pColE1_Alpha2	Kanamycin	#118064
	hCMV-IE1:NLuc	Constitutively expressed NLuc	Transcriptional Unit	pColE1_Alpha2	Kanamycin	#118065
	hCMV-IE1:Renilla/RLuc	Constitutively expressed Renilla/RLuc	Transcriptional Unit	pColE1_Alpha2	Kanamycin	#118066
	hCMV-IE1:GrRenilla/GRLuc	Constitutively expressed GrRenilla/GRLuc	Transcriptional Unit	pColE1_Alpha2	Kanamycin	#118067
Multiplex reporter	MLRV	Multi-luciferase reporter vector	Multigenic vector	pColE1_Alpha2	Kanamycin	#118069

Fig. 3. Schematic overview of vectors required to perform multiplex hextuple luciferase assaying.

(A) Simplified schematics of the constitutively expressing luciferase reporter plasmids for all six luciferases (ELuc, FLuc, RedF, NLuc, Renilla/RLuc, and GrRenilla/GRLuc), that will be used to determine the transmission coefficients (see Fig. 9 and 10), when performing the multiplex hextupe luciferase assay for the first time, or under novel conditions compared to initially established transmission coefficients (culture medium, temperature, and so on). (B) Simplified schematics of six generic pathway transcriptional reporter plasmids included in a generic multiplex hextuple luciferase vector (see C) to perform multiplex pathway analysis using multiplex hextuple luciferase assaying (see Fig. 12). (C) Simplified schematic of the final generic multiplex hextuple luciferase vector consisting of one control and five generic transcriptional luciferase reporter units (see B) stitched together in a specified order to perform multiplex pathway analysis using multiplex hextuple luciferase assaying. (D) A practical example of a final multiplex hextuple luciferase reporter that includes five insulated pathway-responsive luciferase transcriptional units and one constitutively expressed luciferase transcriptional unit used as the control for normalization (see Fig. 12).

In this chapter, we describe how multiplex hextuple luciferase assaying is performed (see Fig. 4). Cultured cells are transfected with a multiplex hextuple luciferase reporter vector, followed by treatment with protein ligands and/or drugs. Alternatively, cells are treated with siRNA first, followed by transfection with a multiplex luciferase reporter vector. The multiplex hextuple vector contains transcriptional reporter units for a control normalization pathway, and five cellular pathways acting upstream of transcription factor-binding motifs, each linked to one of six luciferases: ELuc for the control normalization pathway, and FLuc, RedF, NLuc, Renilla/RLuc, and GrRenilla/GRLuc for the five cellular pathways that are being assayed. Transfected and treated cells are then prepared for multiplex hextuple luciferase assaying. Assaying occurs in a microplate reader equipped with appropriate bandpass filters, BP515-30 and BP530-40 for measuring D-Luciferin-consuming luciferase emissions, and BP410-80 and BP570-100 for measuring CTZ-consuming luciferase emissions. During a first step, three emissions are measured for D-Luciferin-consuming luciferases (ELuc, FLuc, and RedF): total light, BP515-30-filtered light, and BP530-40-filtered light. After quenching the first set of signals, three emissions are measured for the CTZ-consuming luciferases (NLuc, Renilla/RLuc, and GrRenilla/GRLuc): BP410-80-filtered light, BP570-100-filtered light, and total light. Data obtained by the microplate reader for all six readings is then mathematically processed to obtain emission values for each luciferase. Values obtained for the pathway luciferase reporters (FLuc, RedF, NLuc, Renilla/RLuc, and GrRenilla/GRLuc), are then normalized against the control luciferase reporter (ELuc), followed by comparing treated and untreated values, statistical analysis and graphing to visualize potential changes in pathway activities after pharmaceutical or genetic treatment.

Fig. 4. Workflow schematic of multiplex hextuple luciferase assaying.

(A) Cells are transfected with a multiplex hextuple luciferase reporter vector, incubated for 3 h, followed by treatment with protein ligands and/or drugs, and incubated for an additional 24 h (A1), or cells are treated with siRNA first, incubated for 24 h, and then followed by transfection with a multiplex luciferase reporter vector, and incubated for an additional 24 h (A2). Transfected and treated cells are moved to a well of a clear microplate (B), followed by washing (C), and lysis (D). After lysis, the lysate can be stored in a −80 °C freezer to continue the protocol at a later time (Optional). When storage at −80 °C is included, thaw the sample to RT before proceeding with the protocol. (E) Transfer an aliquot of lysate needed for assaying to a white microplate and move microplate to a microplate reader equipped with appropriate bandpass filters (BP515-30 and BP530-40 for measuring D-Luciferin-consuming luciferase emissions, and BP410-80 and BP570-100 for measuring CTZ-consuming luciferase emissions), and add D-Luciferin-containing buffer. (F) Record total light, BP515-30-filtered light, and BP530-40-filtered light, emitted by the D-Luciferin-consuming luciferases (ELuc, FLuc, and RedF). Add quencher- and CTZ-containing solution (G), and record for BP410-80-filtered light, BP570-100-filtered light, and total light, emitted by the CTZ-consuming luciferases (NLuc, Renilla/RLuc, and GrRenilla/GRLuc) (H). (I) Transfer the raw data given by the microplate reader for the D-Luciferin- and CTZ-consuming luciferases to Microsoft Excel software, and mathematically process to obtain emission values for each luciferase. (J) Standardize the luciferase values by dividing values obtained for each of the experimental luciferases (i.e., for FLuc, RedF, NLuc, Renilla/RLuc, and GrRenilla/GRLuc) by the value obtained for the control luciferase (ELuc). (K) Normalize the values by comparing treatment and control values. (L) Perform statistical analysis and graph accordingly to visualize potential changes in pathway activities after pharmaceutical or genetic treatment.

In this chapter, we introduce multiplex hextuple luciferase assaying after genetic perturbation of one out of five cellular signaling pathways each (see Fig. 5). Multiplex hextuple luciferase assaying will allow monitoring the effect of each genetic perturbation on both their target pathway, as well as all other four pathways. Both a generic (see Fig. 5A) and a practical (see Fig. 5B) example are indicated.

Fig. 5. Schematic overview of, genetically perturbing one of five signaling pathways each, that are being probed by multiplex hextuple luciferase assaying.

(A) Simplified schematic of five cellular signaling pathways acting upstream of five transcription factor-binding motifs, each monitored using an orthogonal luciferase: FLuc, RedF, NLuc, Renilla/RLuc and GrRenilla/GRLuc). All perturbations are monitored against a control pathway using ELuc. (B) Practical example of five cellular signaling pathways acting upstream of five transcription factor-binding motifs, each monitored using an orthogonal luciferase: RedF, FLuc Renilla/RLuc, NLuc, and GrRenilla/GRLuc. All perturbations are monitored against a control pathway using the enhanced beetle luciferase reporter (ELuc) (see Fig. 12).

2. Materials

2.1. Living subjects

1. A549 lung cancer cell line.

2.2. Reagents and labware

1. Cell culture medium: Dulbecco's Modified Eagle Medium (DMEM), Nutrient Mixture F-12 (F-12), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) cell culture media.

2. DMEM/F-12+FBS medium: The culture medium plus 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) Penicillin-Streptomycin (P/S, final concentration: 100 U/mL) at 37 °C.

3. 1× PBS, pH 7.2.

4. 1×TrypLE Express Enzyme, phenol red.

5. 0.4% (w/v) Trypan Blue Solution.

6. T75 and T125 flasks.

7. 5 and 10 mL serological pipetes.

8. Clear sterile 96-well microplates for tissue culture, flat bottom.

9. Countess cell counting chamber slide.

10. Lipofectamine RNAiMAX Reagent.

11. siRNAs against transcription factors at a final concentration of 10 nM: GUCACUCUAACGUAUGCAAdTdT against p50 and GAUUGAGGAGAAACGUAAAdTdT against p65 (for the nuclear factor kappa B, NF-κB, pathway), AACAAACCAGGUCUCUUGAUGdTdT against SMAD2 (for the transforming growth factor-β, TGF-β, pathway), GGUCAGAGUCUGGAUCACCdTdT against c-Myc (for the Myc pathway), GAAAUUUGCGUGUGGAGUAdTdT against p53 (for the p53 pathway), and AAGAACGUGACAGAUGAGCAGdTdT against c-Jun, and GAAUUAACCUGGGUGCUGGAdTdT, CUGUCAACGCGCAGGACUUdTdTl, and GGUUCAUUAUUGGAAUUAAdTdT against c-Fos (for the MAPK/JNK pathway).

12. Non-targeting control siRNA: MISSION^® siRNA Universal Negative Control #1.

13. Miniprep prepared multiplex hextuple luciferase reporter vector, quantified by spectrophotometry and verified by agarose gel DNA electrophoresis.

14. 96-well microplate with 25,000 A549 cells/well, seeded the day before the experiment starts (see Section 2.1).

15. Lipofectamine^™ 3000 Transfection Reagent.

16. Opti-MEM I Reduced Serum Medium.

17. Dimethyl sulfoxide (DMSO).

18. Multichannel repeater pipette.

19. Dual-Luciferase^® Reporter Assay System kit, containing 5× Passive Lysis Buffer (5× PLB), Lyophilized Luciferase Assay Substrate, Luciferase Assay Buffer II, 50× Stop & Glo^® Substrate, Stop & Glo^® Buffer.

20. Autoclaved MilliQ water.

21. 5 mL centrifuge tube.

22. CELLSTAR White 384-well microplate.

2.3. Instrumentation

1. Countess automated cell counter.

2. CLARIOstar microplate reader (BMG LABTECH) equipped with appropriate emission bandpass (BP) filters (see Note 1): BP410-80 emission filter measuring light between 370 and 450 nm, BP515-30 emission filter, measuring light between 500 and 530 nm, BP530-40 emission filter, measuring light between 510 and 550 nm, BP570-100 emission filter, measuring light between 520 and 620 nm.

2.4. Software

1. CLARIOstar microplate reader MARS software.

2. Microsoft Excel.

3. GraphPad Prism.

4. Creative Cloud Adobe Illustrator.

3. Methods

3.1. Preparative cell culture work for multiplex hextuple luciferase assaying

1. Thaw a frozen vial of A459 cells on ice and culture the cells in cell culture medium using standard cell culture maintenance techniques.

2. Maintain cells in a total volume of 12.5 mL cell culture medium using a T125 flask. Cells should be passaged twice a week in a 1:8 proportion (see Note 2).

3. One day before multiplex hextuple luciferase assaying, wash the monolayer of cells with PBS for 2 min followed by trypsinization using 3 mL TrypLE Express Enzyme for 5 min at 37 °C.

4. Stop the trypsinization with 5 mL of DMEM/F-12+FBS media.

5. Mix 10 μL of the cells with 10 μL of Trypan Blue Solution, and apply to a Countess cell counting chamber slide.

6. Count the cell number using the Countess automated cell counter.

7. Dilute the cells to 250,000 cells/mL, and dispense 100 μL of cells per well in a tissue culture treated 96-well microplate, resulting in 25,000 cells/well (see Fig. 6, Note 3 and 4). Incubate at 37 °C overnight.

Fig. 6. Microplate schematic illustrating how to seed cells to perform multiplex hextuple luciferase assaying.

Each well of a 96-well microplate that will be tested is seeded with 25,000 cells the day before genetic perturbation: blacks wells indicate wells seeded with cells, while white wells indicate wells, where only culture medium is added (Control).

3.2. RNAi perturbations and transfection of the multiplex hextuple luciferase reporter plasmid

1. Transfect siRNA at a final concentration of 10 nM using Lipofectamine RNAiMAX Reagent, following the microplate configuration as indicated (see Fig. 7).

2. Incubate at 37 °C for 24 h.

3. Dilute all vectors to be transfected to 150 ng/μL.

4. Prepare transfection reaction mixes as follows (see Note 5):

   	1×	Each constitutive luciferase vector (3×)	Multiplex luciferase reporter vector (40×)
   Vector (150 ng/ μL)	1 μL	3 μL	40 μL
   Opti-MEM	15 μL	45 μL	600 μL
   P3000 reagent	0.3 μL	0.9 μL	12 μL
   Lipofectamine reagent	0.3 μL	0.9 μL	12 μL

5. Incubate the transfection reaction mixes for 30 min at room temperature (RT).

6. Add 16.6 μL of the transfection mix per well as indicated (see Fig. 8), ideally using a multichannel repeater pipette to reduce experimental variation.

7. Incubate transfections for 24 h in the cell incubator.

Fig. 7. Microplate schematic illustrating how to perform genetic perturbations of cells before performing multiplex hextuple luciferase assaying.

Transfected cells are treated with RNAi targeting specific transcription factors as indicated: cells treated with RNAi against transcription factor 1, p50/p60 (purple), cells treated with RNAi against transcription factor 2, SMAD2 (pink), cells treated with RNAi against transcription factor 3, c-Myc (green), cells treated with RNAi against transcription factor 4, p53 (teal), cells treated with RNAi against transcription factor 5, c-Jun/c-Fos (light brown), and cells treated with a control RNAi (dark brown). Untreated cells (black wells) will be used to determine transmission coefficients.

Fig. 8. Microplate schematic illustrating how to transfect cells before performing multiplex hextuple luciferase assaying.

Wells seeded with cells (see Fig. 6) are transfected with plasmid sample: black wells are transfected with the multiplex hextuple luciferase reporter vector (M) containing five pathway transcriptional luciferase reporters (FLuc, RedF, NLuc, Renilla/RLuc, and GrRenilla/GRLuc) and one control luciferase reporter (ELuc), used to measure pathway-specific manipulations (see Fig. 12)., while dark green, pink, purple, teal, green, and light brown wells are transfected with constitutively expressing ELuc (E), FLuc (F), RedF (RF), NLuc (NL), Renilla/RLuc (Re), or GrRenilla//GRLuc (GR) luciferase reporter vectors, respectively, used to determine the transmission coefficients (see Fig. 9 and 10).

3.3. Multiplex hextuple luciferase assaying

1. Thaw Luciferase Assay Buffer II and the Stop & Glo^® Buffer.

2. Prepare the Luciferase Assay Reagent II (LAR II) buffer by resuspending the lyophilized Luciferase Assay Substrate with the Luciferase Assay Buffer II. Make 5 mL aliquots of LAR II buffer and store aliquots at −80 °C (see Note 6).

3. Make 5 mL aliquots of the Stop & Glo^® Buffer, but don’t mix it with the Stop & Glo^® substrate (see Note 7).

4. Prepare 2 mL of 1× Passive Lysis Buffer (1× PLB) by mixing 400 μL of the 5× PLB with 1.6 mL of MiliQ water (see Note 8).

5. Take the 96-well microplate from the incubator, and remove the culture media with a multichannel repeater pipette (see Note 9).

6. Wash the wells once with 150 μL of PBS during 1 min. Remove the PBS with a multichannel repeater pipette.

7. Add 40 μL of 1× PLB, and incubate at RT for 30 min. Lysates do not need to be cleared by centrifugation (see Notes 10 and 11).

8. Dilute 30 μL of the Stop & Glo^® Substrate (50×) in 1470 μL of Stop & Glo^® Buffer to generate 1.5 mL of Stop & Glo^® Reagent (see Note 12).

9. Generate a multiplex luciferase protocol using the CLARIOstar microplate reader MARS software with the following settings:

   1. Dispense 10 μL of LARII buffer.
   2. Wait 30 s.
   3. Measure the total light for 2 s.
   4. Measure BP515-30 filtered light for 2 s.
   5. Measure BP530-40 filtered light for 2 s.
   6. Dispense 15 μL of the Stop & Glo^® Reagent.
   7. Wait 7 s.
   8. Measure BP410-80 filtered light for 1 s.
   9. Measure BP570-100 filtered light for 1 s.
   10. Measure total light for 1 s.

10. Load injector 1 with at least 1.5 mL LARII buffer and injector 2 with at least 1.5 mL Stop & Glo^® Reagent (see Note 12).

11. Thaw the cell lysates of the samples with the constitutively expressing luciferases if these had been frozen at −80 °C.

12. Briefly spin down in centrifuge to collect at the bottom of the well.

13. Transfer 5 μL of the cell lysates to a white 384-well microplate for the constitutive luciferases, make four technical replicates, and insert the microplate into the CLARIOStar luminometer.

14. Start the multiplex luciferase protocol using the CLARIOstar software, and export the recorded data.

15. Use the recorded values to calculate the transmission coefficients (κ) for each luciferase as shown schematically (see Figs. 9 and 10).

16. Thaw the lysates of the samples transfected with the multiplex hextuple luciferase vectors if these had been frozen at −80 °C. Briefly spin down in centrifuge to collect at the bottom of the well.

17. Transfer 40 μL of the lysates to a white 384-well microplate, and insert the microplate into the CLARIOStar microplate reader.

18. Start the multiplex luciferase protocol using the CLARIOstar MARS software, and export the recorded data to Microsoft Excel.

19. Use obtained values to calculate the individual contribution of each luciferase to the mix as shown schematically (see Fig. 11).

20. Standardize all luciferase measurements in each sample by dividing the values of the FLuc, RedF, NLuc, Renilla/RLuc and GrRenilla/GRLuc luciferases by the value obtained for ELuc.

    $$\begin{gathered} \mathbf{NF}\text{-}\kappa \mathbf{B}\mathbf{expression}=\frac{\text{RedF value}}{\text{ELuc value}} \\ \mathbf{TGF}\text{-}\beta \mathbf{expression}=\frac{\text{FLuc value}}{\text{ELuc value}} \\ \mathbf{c}\text{-}\mathbf{MYC}\mathbf{expression}=\frac{\text{Renilla}∕\text{RLuc value}}{\text{ELuc value}} \\ \mathbf{p}53\mathbf{expression}=\frac{\text{NLuc value}}{\text{ELuc value}} \\ \mathbf{MAPK}∕\mathbf{JNK}\mathbf{expression}=\frac{\text{GrRenilla}∕\text{GRLuc value}}{\text{ELuc value}} \end{gathered}$$

21. Calculate the mean for each data set, and the standard error of the mean (SEM).

22. For each pathway, normalize each treatment mean and SEM by dividing them by the value of the control.

23. Calculate the log2 fold-change, by doing the base 2 logarithm of the normalized values and propagate the errors, according to the formula:

    $${\mathbf{Log}}_2(\mathbf{N}\pm \mathbf{SEM})={\text{Log}}_2\mathrm{N}\pm \frac{1}{\text{Ln}(2)}\frac{\text{SEM}}{\mathrm{N}}$$

24. Determine the statistical significance of the log2 fold-change by the multiple t-test using the Holm-Sidak method with alpha = 0.05 (*P <0.05, **P < 0.01, ***P < 0.001, and ****P < 0001, n.s. is non-significant), referred to the control values for each pathway.

25. Represent the values in bar graphs, heat maps or boxes & whiskers, according to your preferences and preferred software. Results of the experiments detailed in this chapter were plotted using the GraphPad Prism software for statistical analysis and graphing, followed by Creative Cloud Adobe Illustrator for illustration (see Fig. 12).

Fig. 9. Schematic overview illustrating how to calculate transmission coefficients for the three D-Luciferin-consuming luciferases, as well as the three CTZ-consuming luciferases.

(A) Simplified schematic of the constitutively expressing luciferase reporter plasmids for all six luciferases (ELuc, FLuc, RedF, NLuc, Renilla/RLuc, and GrRenilla/GRLuc), used to determine the transmission coefficients (see also Fig. 3): (B) Emission spectra of the three D-Luciferin-consuming luciferases used in the multiplex hextuple luciferase assay. Two bandpass emission filters, one measuring between 500 and 530 nm (BP515-30), and a second measuring between 510 and 550 nm (BP530-40), are used to capture select portions of the three emission spectra that will be used for spectral unmixing (see Fig. 11). (C) Emission spectra of the three CTZ-consuming luciferases used in the multiplex hextuple luciferase assay. Two additional bandpass emission filters, one measuring between 370 and 450 nm (BP410-80), and a second measuring between 520 and 620 nm (BP570-100), are used to capture select portions of the emission spectrum that will be used for spectral unmixing (see Fig. 11). (D) Simplified schematic of the experimental setup to determine transmission coefficients for all six luciferases using either total BL emission or bandpass filtered emission over the indicated filters. (E) Formulas to calculate the transmission coefficients (κ) of each D-Luciferin-consuming luciferase over the indicated bandpass emission filters. (F) Formulas to calculate the transmission coefficients (κ) of each CTZ-consuming luciferase over the indicated bandpass emission filters.

Fig. 10. Calculating the transmission coefficients for the three D-Luciferin-consuming luciferases, as well as the three CTZ-consuming luciferases.

(A) Calculation of the transmission coefficients for the three D-Luciferin-consuming luciferases: κELuc₅₁₅, κFLuc₅₁₅, and κRedF₅₁₅ represent the transmission coefficients over the BP515-30 bandpass emission filter (left), while κELuc₅₃₀, κFLuc₅₃₀, and κRedF₅₃₀ represent the transmission coefficients over the BP530-40 bandpass emission filter (right). (B) Calculation of the transmission coefficients for the three CTZ-responsive luciferases, κNLuc₄₁₀, κRenilla/RLuc₄₁₀, and κGrRenilla/GRLuc₄₁₀ represent the transmission coefficients over the BP410-80 bandpass emission filter (left), while κNLuc₅₇₀, κRenilla/RLuc ₅₇₀, and κGrRenilla/GRLuc ₅₇₀ represent the transmission coefficients over the BP570-100 bandpass emission filter (right).

Fig. 11. Calculating luciferase-specific emission contributions in a mixture of three D-Luciferin-consuming luciferases, or three CTZ-consuming luciferases, using previously determined transmission coefficients and simultaneous equations.

(A) Simultaneous equations being used to solve the D-Luciferin-consuming luciferase contributions in a mixture have three unknown variables corresponding to the amount of light that is specifically emitted by each D-Luciferin-consuming luciferase (ELuc, FLuc, and RedF). (B) Simultaneous equations being used to solve the CTZ-consuming luciferase contributions in a mixture have three unknown variables corresponding to the amount of light that is specifically emitted by each CTZ-consuming luciferase (NLuc, Renilla/RLuc, and GrRenilla/GRLuc). (C) To obtain calculated values for each D-Luciferin-consuming luciferase-linked reporter unit, a matrix inversion of the coefficient matrix (the matrix containing values for all transmission coefficients) obtained using the appropriate bandpass emission filters (see Fig. 10), is multiplied by the value matrix (the matrix containing BL measurements obtained by the microplate reader). (D) Similarly, to obtain calculated values for each CTZ-consuming luciferase-linked reporter unit, a matrix inversion of the coefficient matrix (the matrix containing values for all transmission coefficients) obtained using the appropriate bandpass emission filters (see Fig. 10), is multiplied by the value matrix (the matrix containing BL measurements obtained by the microplate reader).

Fig. 12. Multiplex hextuple luciferase assaying detects both on-target and off-target effects while genetically perturbing cellular signaling upstream of a specific transcription factor-binding motif.

(A) Simplified schematic of five cellular signaling pathways acting upstream of five transcription factor-binding motifs, each monitored using an orthogonal luciferase: RedF, FLuc, Renilla/RLuc, NLuc and GrRenilla/GRLuc. All perturbations are monitored against a control pathway using the enhanced beetle luciferase reporter (ELuc). (B) The final multi-luciferase plasmid includes five insulated pathway-responsive luciferase transcriptional units and one constitutively expressed luciferase transcriptional unit used as the control for normalization. (C-G) On-target downregulation of a signaling pathway after adding siRNA(s) against (a) key transcription factor(s) binding a transcription factor-binding motif for the NF-κβ (C), TGF-β (D) c-Myc (E), p53 (F), or MAPK/JNK pathway (G). Off-targeting effects are indicated as well. Statistical significance of the fold-change of different genes analyzed by pathways in the multiplex luciferase assay was determined by multiple t-tests using the Holm-Sidak method with alpha = 0.05 (*P <0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, n.s. is non-significant). n=4 for all multiplex hextuple luciferase assays.

3.4. Time considerations for the entire procedure

To ensure that cells are healthy, maintenance of the A549 cell line should start two weeks before the multiplex hextuple luciferase assaying. Once cells are ready, the complete procedure can be performed in a single work week (see Fig. 13). On day 1, count and plate A549 cells. On day 3, treat cells with siRNA(s), and 24 h later, transfect the multiplex hextuple luciferase reporter vector and the control luciferase plasmids. On day 5, wash and lyse the cells, and calibrate the luminometer with the six constitutively expressed luciferase vectors. After calibration, perform multiplex hextuple luciferase assaying, and analyze and plot the data.

Fig. 13. Experimental timeline.

Gantt chart illustrating tasks and milestones for the different protocols, as well as working days across one calendar weeks (Day 1 to Day 5).

4. Notes

1. While our initial work developing and implementing the multiplex luciferase assay, as well as this work, is all performed using the CLARIOstar microplate reader and emission filters from BMG LABTECH, any microplate reader equipped with appropriate emission bandpass filters should be able to perform the needed measurements, after appropriate calibration.

2. Do not maintain your cultures for more than 30 passages. Cells should be passaged twice a week in a 1:8 proportion.

3. Prepare 4 to 6 wells per experimental condition using the multiplex hextuple luciferase assay. Prepare also two wells for each constitutive luciferase control. We prepared 48 wells for the experiment indicated (see Fig. 6).

4. The number of cells is optimized for the A459 cell line. When using other cell lines, the number of cells per well may need to be adjusted accordingly: difficult to transfect cells may need more cells to obtain decent enough luciferase emission signals.

5. Multiply the amount of the transfection mix by the number of biological replicates and add at least 10%. For the experiments in this chapter, we needed to transfect 36 wells of the multiplex luciferase reporter vector, and 2 of each control vector, so we prepared a master mix consisting of 40× and 3× of the reaction mix for each vector, respectively.

6. Repeated freeze-thaw cycles will decrease assay performance. Also, aliquots are less stable at −20 °C, so we recommend storing 2 or 3 at −20 °C for short term storage.

7. Stop & Glo^® Buffer mixed with Stop & Glo^® substrate (Stop & Glo^® Reagent) should be prepared just before each use. Both reagents are stable at −20 °C but once it is mixed with the substrate (Stop & Glo^® Reagent), activity decreases in a few days.

8. We recommend a minimum of 40 μL 1× PLB per well in a 96-well microplate. For our experiment, totaling 48 wells, we need at least 1.92 mL.

9. Do not use a vacuum aspirator. This will disturb the cells.

10. After lysis, we recommend a 2 h freeze at −80 °C for improved lysis performance.

11. Optional protocol stopping point. You can store the lysate at −80 °C for up to a month for later luciferase measurements.

12. The minimum suggested reagent, for both LARII and Stop & Glo^® Reagent, per injector in the CLARIOStar is 1.5 mL, regardless of the amount needed to ensure correct injector priming. For our experiment, totaling 48 wells, we need at least 0.48 mL (10 μL/well) and 0.72 mL (15 μL/well) of LARII and Stop & Glo^® Reagent, respectively.