Skip to main content
. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Curr Protoc Mol Biol. 2020 Jun;131(1):e122. doi: 10.1002/cpmb.122

Figure 1. Workflow schematic of multiplex hextuple luciferase assaying.

Figure 1.

(A) Transfect a test sample of cells with a multiplex luciferase reporter vector and incubate for 3 hours. The multiplex vector contains transcriptional reporter units for specific cellular pathways, each linked to one of six luciferases: ELuc (E), Flux (F), RedF (RF), NLuc (N), Renilla (Re), and GrRenilla (GR). (B) Treat cells with appropriate ligands and/or drugs, and incubate for an additional 24 hours. (C) Move transfected and treated cells to a well of a clear microtiter plate. (D) Wash cells with 150 μl PBS buffer. (E) Lyse cells for 30 minutes with 40 μl PLB1X lysis buffer. Optional: Store lysate in a −80°C freezer to continue the protocol at a later time point. When this step is included, thaw the sample to room temperature before proceeding to the next step. (F) Transfer an aliquot of lysate to a white microtiter plate and move plate to a plate reader equipped with appropriate bandpass filters, i.e., BP515–30 and BP530–40 for measuring D-Luciferin-consuming luciferase emissions, and BP410–80 and BP570–100 for measuring coelenterazine-luciferase emissions, and add 10 μl of D-Luciferin-containing LARII buffer. (G) Wait 30 seconds and record for 2 seconds each, total light, BP515–30-filtered light, and BP530–40-filtered light, emitted by the D-Luciferin-luciferases (ELuc, FLuc, and RedF). (H) Add 15 μl of quencher- and coelenterazine-containing Stop & Glo Reagent. (I) Wait 7 seconds and record for, 1 second each, BP410–80-filtered light, BP570–100-filtered light, and total light, emitted by the coelenterazine-luciferases (NLuc, Renilla, and GrRenilla). (J) Transfer the raw data given by the plate reader for the D-Luciferin and coelenterazine luciferases to the provided Excel template to calculate emission values for each luciferase. (K) Standardize the luciferase values by dividing values obtained for each of the experimental luciferases (i.e., for FLuc, RedF, NLuc, Renilla, and GrRenilla) by the value obtained for the control luciferase (ELuc). (L) Normalize the values by comparing treatment and control values. (M) Perform statistical analysis and graph accordingly.