Multiplex Hextuple Luciferase Assaying

Abstract

We recently expanded the commonly used dual luciferase assaying method toward 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 detailed protocols on how to perform such multiplex hextuple luciferase assaying to monitor cellular signal processing upstream of five transcription 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 toward 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 appropriately 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.

Keywords: Assay; Cell culture; Cellular signaling pathway; Hextuple; Luciferase; Microplate reader; Multiplex; Orthogonal; Pathway perturbation; Transfection.

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.

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.

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).

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.

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).

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).

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).

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.

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).