Establishing a multiplex hextuple luciferase assay to examine multiple cellular pathways at once

Introduction

Most cell-based assays rely on a single measurement yielding limited information about the biological system being studied. Multiplexed reporter assays seek to address this limitation. Particularly, luciferases have advantages for multiplexing over fluorescent proteins, including higher sensitivity, wider dynamic range and the absence of auto-luminescence. The most common multiplex luciferase assay (the dual reporter assay) uses sequential detection of two luciferases with distinct substrate specificity and the ability to quench the first luciferase. Here we describe how the expanding palette of 
luciferases can now be employed such that 6 signals may be discriminated by adding spectral deconvolution.

Assay Principle

The essential aspects of the multiplex hextuple luciferase assay are shown in Figure 1.

One vector encoding six cellular signaling reporters, each associated with a distinct luciferase is solotransfected into cells. After incubation to allow luciferase expression, cells can be treated and total luciferase signals, or filtered light, detected for 3 D-Luciferin (D-LUC)-consuming luciferases followed by quenching and similar detection of 3 coelenterazine (CLZN)-consuming luciferases. Overlapping spectra are deconvoluted through calculation so that the relative expression for each signaling reporter can be found.

Materials & Methods

• Cellstar white, 384-well microplates (Greiner Bio-One)
• Dual Luciferase^® Reporter (DLR^TM) Assay (Promega)
• CLARIOstar^®
• Luciferases (Addgene)

Experimental Procedure
Plasmid preparation, cell culture and plasmid transfection were performed as described^1-5. Luciferase emissions were obtained using DLR^TM reagents 24 hours after transfection with or without treatments. RNAi was performed for 24 hours before transfection. Cells were washed with PBS and lysed with Passive Lysis Buffer and 5 µl of lysate was transferred to microplates for data collection.

Measurement of Unique Bioluminescent Spectra

Luciferase measurements were initiated using LARII buffer for D-LUC-consuming luciferases or LARII and Stop & Glo^® buffer for CLZN-consuming luciferases. They were read on a CLARIOstar using spectral scanning for each luciferase individually first to find cohorts that could be distinguished from one another while using the same substrate.

Instrument Settings

Measurement of Multiplex Bioluminescent Spectra
Luciferase measurements were obtained on a CLARIOstar using LARII buffer for D-Luc turnover then Stop & Glo^® buffer injections and the following settings for D-LUC- and CLZN-consuming luciferases.

Results & Discussion

Twelve luciferases were characterized to determine the ability to spectrally resolve multiple signals. Figure 2 shows the results for 6 D-LUC- (Left) and 6 CLZN-consuming luciferases (Right)¹Based on the results of the above spectral data as well as substrate specificity and quenching capacity1, 6 luciferases were brought forward for further testing (Table 1).

Table 1: Luciferases used in hextuple multiplexing (* results indicate that CLZN is a suitable substrate for NLuc)¹

These 2 groups of 3 luciferases each were each further characterized to ensure that the contribution of each luciferase could be deconvoluted from the light produced by all, based on the filters shown in instrument settings and mathematical calculations¹. In addition, assessment of signal stability showed that the delay after injection, multichromatic order and integration measurement time in the instrument settings are suitable¹. Furthermore, experiments showed that the experimental approach employed allows for accurate quantification of static and dynamic levels of each luciferase in a mixture¹.

Proof of principle testing was performed to detect direct and collateral effects of knocking down a single constituent of the multiplex luciferase assay by proven siRNA approaches (Figure 3). Besides target knockdown, clear effects on the expression of other constituents may be observed.

Conclusion