Measuring luciferase expression using the SpectraMax Glo Steady-Luc Reporter Assay Kit

Luciferase standard curve

To identify the linear detection range of this assay, a standard curve was constructed to measure the RLU as a function of luciferase concentration. All components of the SpectraMax Glo Steady-Luc Reporter Assay Kit were first allowed to equilibrate to room temperature. SpectraMax Glo Steady-Luc working solution was made by adding D-Luciferin to the Steady-Luc Assay buffer in a 1 mg to 4 mL ratio. A ten-fold serial dilution of purified luciferase in PBS with 0.01% BSA starting at a concentration of 1 x 10⁶fg/well was prepared, and 25 μL of each concentration was added in triplicate to a 384-well solid white plate. Afterwards, 25 μL SpectraMax Glo Steady-Luc working solution was added to the wells containing luciferase and controls. The plate was shaken and incubated in the dark for 10 minutes. Using the SpectraMax i3x reader, the preconfigured SpectraMax Glo Steady-Luc Reporter assay protocol in SoftMax Pro Software was used to measure luminescence. After detection, the protocol automatically generates a curve of the data.

Transfection and cell-dilution assay

Part of assay development for a reporter gene assay is to optimize the cell number and amount of plasmid necessary to give a robust signal for screening. A cell dilution assay was performed with two different amounts of pGL4.13 firefly luciferase starting in 6-well plates. CHO-K1 cells were transiently transfected with 1.7 μg or 0.8 μg per well of pGL4.13[luc2/SV40] vector, which encodes the luciferase gene luc2 under control of the SV40 early enhancer/promoter, or pGL3-Basic (control) vector, which lacked promoter and enhancer sequences. After 24 hours, cells were trypsinized and seeded at 100 μL/well into a 96-well plate starting at 30,000 cells/well with subsequent 2-fold dilutions. 100 μL/well Steady-Luc working solution was added to the wells. The plate was covered to protect the reagents from light and mixed using an orbital shaker. After 10 minutes, luminescence was measured and recorded on the SpectraMax i3x reader using the preconfigured SpectraMax Glo Steady-Luc Reporter assay protocol. The preconfigured protocol is found in the Reporter Assays section under the Protocol Library Tab or on the SoftMax Pro Software protocol sharing website (www.softmaxpro.org).

Screening applications

A reporter gene assay screen was simulated by performing multiple reads of a plate of pGl4 firefly luciferase transfected cells using the SpectraMax Glo Steady-Luc Reporter Assay Kit. Cells were transfected as previously described and plated at 30,000 cells per well in a 96-well plate and grown overnight. On the day of the assay, the plate was equilibrated to room temperate, and then 100 μL reconstituted Steady-Luc working solution containing D- Luciferin was added to each well. The plate was covered and mixed on an orbital shaker for five minutes and then placed In a SpectraMax i3x reader and mixed. Luminescence was read every five minutes for a total of five hours with three seconds of orbital shaking before each read.