Luciferase Reporter Gene Assay Kit, BioAssay™

The Luciferase Reporter Gene Assay is based on the quantitation of luciferase expression in mammalian, yeast or E. coil cells, using luciferin and ATP as substrates. The reaction results in light production which can be conveniently measured on a luminometer.|Reaction scheme:|Luciferase|ATP+D-luciferin+O2 oxyluciferin+AMP+PPi+CO2+light|This bioluminescent reporter gene assay is extremely sensitive and is especially suitable for quantifying luciferase expression in recombinant cells. This ultra-sensitive, homogeneous cell-based assay only requires adding a single reagent to the cells and measuring the light intensity after a short incubation step (2 minutes). Assays can be performed in tubes, cuvettes or multi-well plates. All kit components are compatible with culture media and with all liquid handling systems. With an extended luminescence emission kinetics (half-life 40 min), the luciferase assays are especially suitable for high-throughput screening in 96-well, 384-well and 1536-well plates. In addition, the reagent provided in the kits has been formulated for maximum sensitivity, reproducibility and long shelf-life.||Applications:|Gene Regulation: gene expression level, characterization of promoter activity, modulation of gene expression by receptors, transcription factors and small molecules.|Drug Discovery: high-throughput screen for gene modulators.||Key Features:|High sensitivity and wide detection range: detection of as little of 2fg luciferase and as few as 4 cells. Plus, the emitted light is linear over seven orders of magnitude.|Compatible with routine laboratory and HTS formats: assays can be performed in tubes or microplates, on LJL Analyst, Berthold Luminometer, Top-Count, MicroBeta counters, chemiluminescent image plate readers (CLIPR/LeadSeeker). Assay reagents compatible with all liquid handling systems.|Fast and convenient: homogeneous “mix-and-measure” assay allows detection of luciferase levels within 10 minutes. The optimally combined reagent system allows a single addition step, and simultaneous cell lysis and detection.|Robust and amenable to HTS: Z’ factors of 0.6 to 0.8 are observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems.||Kit Contents:|Reagent Buffer|Assay Buffer||Storage Conditions:|Store the Reagent in the provided amber tube at -20°C and the Assay Buffer at 2-8°C. Shelf life: 12 months after receipt.|Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|The Luciferase Reporter Gene Assay is based on the bioluminescence generated during the luciferin/luciferase reaction. The reconstituted reagent has been optimized to combine cell lysis and detection into one single step. Phenol red in culture media does not interfere in this assay. All data in the Technical Notes: were obtained in media containing phenol red.||Important: fresh reconstitution of the Reagent in Assay Buffer is recommended, although the reconstituted Reagent may be stable for up to 4 weeks when stored at -20°C.||Procedure using 96-well plate:|1. Plate and culture cells (80ul) in white opaque 96-well tissue culture plates. Typical culture medium contains DMEM, 10% fetal bovine serum and antibiotics (penicillin/ streptomycin, gentamycin, etc). Amino acids and other nutrients can be added to the culture medium. Assays can be performed on either adherent cells or cells in suspension. The cells can be either stably or transiently transfected with the luciferase gene. Culture volume can vary from 50 to 100ul, although 80ul is used in this protocol. Blank control wells containing no cells should also be prepared.|2. Add test compounds and controls to cells. Mix well and incubate for the cells desired period of time. Incubation time for gene regulation studies can be from several hours up to 3 days. It is recommended that assays be run in duplicate or triplicate. A volume of 20ul compounds in PBS or culture medium is recommended.|3. Reconstitute the Reagent. First equilibrate the Reagent and Assay Buffer to room temperature. Then simply combine the Assay Buffer and Reagent by pipetting a small volume (e.g. 1ml) buffer to the Reagent tube. Vortex briefly and pipet the reconstituted solution to the Assay Buffer bottle. Repeat this step to transfer all Reagent to the Assay Buffer bottle. Mix by inversion until the Reagent is thoroughly dissolved. After this is done, mark the bottle label as Reconstituted Reagent.|4. Add 100ul (per 80ul of cell culture) of the reconstituted Reagent to each well and mix well with the cells. Incubate for 2 minutes at room temperature. The volume of the reagent can be adjusted depending on the volume of cell culture.|5. Measure luminescence on a luminometer. The integration time can be 1 sec to 2 min depending on the luciferase expression level and instrument sensitivity. For most luminometers (Berthold Luminometer, LJL Analyst, Top Count, MicroBeta Counters, CLIPR and LeadSeeker), integration 1 to 5 sec is appropriate.|Procedure using 384-well plate:|1. Plate and culture cells (25ul) in white opaque 384-well tissue culture plates. Culture volume can vary from 20 to 50ul, although 25ul is used in this protocol. Set up blank control wells containing no cells.|2. Add test compounds and controls to cells. Mix well and incubate for the cells desired period of time. A volume of 5ul compounds in PBS or culture medium is recommended.|3. Reconstitute the Reagent using the same procedure as for the 96-well assay.|4. Add 30ul (per 25ul of cell culture) of the reconstituted Reagent per well and mix well with the cells. Incubate for 2 minutes at room temperature. The volume of the reagent can be adjusted depending on the volume of cell culture.|5. Measure luminescence on a luminometer. The integration time can be 1 sec to 2 min depending on the luciferase expression level and instrument sensitivity. For most luminometers (Berthold Luminometer, LJL Analyst, Top Count, MicroBeta Counters, CLIPR and LeadSeeker), integration 1 to 5 sec is appropriate.||General Considerations:|Incubation time. Both the luciferin/luciferase reaction and cell lysis are fast, so incubation for 2 to 10 minutes following reagent addition is generally enough for mammalian cells (e.g. HEK293, CHO). |Cell number. The optimized reporter gene assay reagent is very sensitive to luciferase (detection limit 2 fg) and exhibits linearity over seven orders of magnitude. As few as 4 cells can be determined and a linear response is still observed with as many as 80,000 cells per 96- well. For assay optimization, it is recommended that the optimal number of cells per well be determined by serial dilution of cells. Cells can be adherent or in suspension cultures.|Cell lysis and mixing. For the sake of convenience, the addition of 1 volume of reconstituted reagent to 1 volume of cells allows a sufficient mixing. No additional mixing is required since the specially formulated buffer instantly lyses mammalian cells.||Data Analysis:|The light intensity (RLU) is directly proportional to the luciferase concentration. For dose-response studies, the data are plotted against compound concentration and the EC50 for gene up-regulator compound and IC50 for a gene down regulator compound can be determined by non-linear regression analysis using Prism or other Data Analysis: tools.||Technical Notes:|The Luciferase Reporter Gene Assay Kit has been specially optimized and formulated to provide a sensitive, convenient and robust assay for gene expression and regulation studies in mammalian cells.|Key Features: of the kits are as follows:|High sensitivity and wide detection range: detection of as little of 2 fg luciferase and as few as 4 cells. Plus, the emitted light is linear over seven orders of magnitude.|Compatible with routine laboratory and HTS formats: assays can be performed in tubes or microplates, on LJL Analyst, Berthold Luminometer, Top-Count, MicroBeta counters, chemiluminescent image plate readers (CLIPR/LeadSeeker). Assay reagents compatible with all liquid handling systems.|Fast and convenient: homogeneous “mix-and-measure” assay allows detection of luciferase levels within 10 minutes. The optimally combined reagent system allows a single addition step and simultaneous cell lysis and detection.|Robust and amenable to HTS. Z’ factors of 0.7 to 0.9 are routinely observed in 96-well and 384-well plates. Can be readily automated on HTS liquid handling systems.