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HOME > Protocols > Cell Biology > Luminometer Protocol for Bright-GloÖ

Luminometer Protocol for Bright-GloÖ

1. INTRODUCTION

The Reporter™Microplate Luminometer, combined with Promega’s Bright-Glo™ Luciferase Assay System, provides an easy to use, sensitive method in the quantitation of gene expression. Transcriptional regulation, coupled to the expression of a reporter gene, is regularly used to study a wide range of physiological events. A popular example is the analysis of receptor function by quantifying the action of specific receptor response elements on gene expression. Luciferase is a recommended choice as a reporter because functional enzyme is created immediately upon translation, and the assay is swift, dependable and easy to perform1, 2. Additionally, analysis using luciferase as the genetic reporter is appropriate for laboratory automation and high-throughput applications. For these reasons, luciferase is widely used in the pharmaceutical and biotechnology industries.

The Bright-Glo™ Luciferase Assay System from Promega Corporation has been developed specifically to maximize the sensitivity of the assay reagent while providing a luminescent signal half-life generally exceeding 25 minutes.

Broad applicability of luciferase reagents is necessary for wide use in homogenous, single-step assays and in nonhomogeneous, two-step assays. The Bright-Glo™ reagent is compatible with most commonly used culture media for mammalian cells (RPMI 1640, MEM?, DMEM and F12, with or without added serum), and can tolerate phenol red and organic solvents. Furthermore, the reagent is tolerant of incomplete mixing, making it suitable for multiwell plates.

2. MATERIALS REQUIRED

  • The Reporter™ Microplate Luminometer (Turner BioSystems P/N 9600-001)
  • Bright-Glo™ Luciferase Assay System (Promega Catalog #'s E2610, E2620, E2650)
  • 8 channel 200m l Autopipettor
  • 200m l Pipette Tips
  • Solid White Assay Plate, 96-well, high binding, flat (VWR P/N 29444-020)

3. REAGENT PREPARATION

3.1 Bright-Glo™ Substrate: Use as supplied. Store at -20░ C, where it is stable for up to 6 months. The substrate may also be stored at 4░ C for up to two weeks.

3.2 Bright-Glo™ Buffer: Use as supplied. Store below 25░ C.

3.3 Bright-Glo™ Reagent: Transfer the contents of one bottle of Bright-Glo™ Buffer to one bottle of Bright-Glo™ Substrate. Mix by inversion until the substrate is thoroughly dissolved. Use reconstituted reagent on the same day of its preparation or store at –70░ C for up to one month.

Note: The temperature of the Bright-Gloń Reagent should be held constant at room temperature while quantitating luminescence since luciferase activity is temperature dependent. Reagent stored frozen after reconstitution must be thawed below 25░ C to ensure reagent performance. Mix well after thawing. The simplest method for thawing is placing the reagent in a water bath at room temperature.

4.0 Instrument Set-up

4.1 Turn on both the Reporter™ Microplate Luminometer and the computer. Allow the instrument to warm up for at least 10 minutes.

4.2 Double click on the Reporter icon to start the software.

4.3 In the upper left corner, click on ‘File’ then ‘New’.

4.4 Enter your information into the ‘Test’, ‘Operator’, ‘Plate No.’, and ‘Notes’ fields, as desired.

4.5 Click on the ‘Options’ button to open the options screen

4.6 Select ‘Single Run’ or ‘Multiple Run’ as desired.

4.7 Click on each well that you want to be read. Wells selected will be green. Unselected wells will be white. To reduce the reading time, select only the wells that will have the Bright-Glo reagent.

4.8 Click ‘OK’ when selections are completed.

5.0 Sample Analysis

5.1 Remove 96 well plate containing cell cultures from the incubator. Note: For maximum reproducibility, equilibrate cell cultures to room temperature before adding reagent.

5.2 Add a volume of the Bright-Gloń reagent equal to that of the culture medium in each well. For 96 well plates, typically 100 ul of reagent is added to cells grown in 100 ul of medium.

5.3 Press sample compartment door button on the front of the Reporter™ Microplate Luminometer to open the sample compartment.

5.4 Carefully insert 96 well plate with samples and reagent into the tray. Avoid splashing or spilling samples.

5.5 Press button to close door.

5.6 Wait at least 2 minutes to allow for complete cell lysis. However, for maximal light intensity, samples should be analyzed within 15 minutes of reagent addition.

5.7 Click on ‘Start’ to begin sample readings. You will be prompted to enter a file name for the data file. Enter a file name and click on ‘Save’. After you click on ‘Save’, the run will begin.

5.8 Remove 96 well plate from the instrument when measurements are complete and data are displayed on the main screen.

6. REFERENCES

1 Ow, D.W. et al. (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 234, 856.

2 De wet, J.R. et al. (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725

CAUTION:

The lyophilized Bright-Glo® Substrate contains dithiothreitol (DTT) and is therefore classified as hazardous. The reconstituted reagent is not known to present any hazards as the concentration of DTT is less than 1%. However, we recommend the use of gloves, lab coats and eye protection when working with these or any chemical reagents. Promega and Turner BioSystems assume no liability for damage resulting from handling or contact with these products.

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