( Evelyn McGown Ph.D. Michael Su...)Luciferase Reporter Gene Cells and Recombinant Luciferase Dilutions Studied with the CLIPR Luciferase Assay Kit and the LMax Microplate Luminometer (MaxLine Application Note #38)

Evelyn McGown, Ph.D. , Michael Su, M.S. and Jin Liao, M.D., Ph.D.
Molecular Devices Corporation, 8/00

INTRODUCTION
Luciferase from the North American firefly (Photinus pyralis) is very popular foruse in Reporter Gene Assays. The enzyme catalyzes the ATP-dependent oxidationof luciferin with the concomitant release of light (Figure 1)¹. When luciferase is the limiting component in the reaction, the amount of light emitted isproportional to the concentration of luciferase. The reaction is extremely efficientand the quantum yield is the highest of any characterized bioluminescentreaction. The bright signal makes this a valuable enzyme to use for reportingpromoter activity. The intensity of the emitted light can be measured easily withan Lmax microplate luminometer.

The CLIPRLuciferase Assay reagent was originally developed for use in aglow kinetics mode in the Molecular Devices CLIPR high throughputmicroplate luminometer. However, the reagent can also be used in a flash modeto measure cell-based luciferase in Molecular Devices Lmax microplate luminometer. Cell lysis and enzyme detection occur upon addition of the single reagent: no pre-lysis or other pre-treatment step is necessary. Results are presented below.

MATERIALS
1. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)

2. Recombinant Firefly Luciferase, Sigma Cat # 9506.

3. CLIPR Luciferase Assay Kit, Cat. No. R 8032; Molecular Devices Corporation; 1-800-635-5577. The kit contains luciferin (lyophilized) and luciferase assay buffer.

4. PBS 1X - Dulbeccos Phosphate Buffered Saline (PBS). Irvine Scientific, Cat. #9235. Phone: 800-437-5706.

5. Solid white 96-well microplates; e.g. CorningCostar Cat. No. 3912, Tel: 1 8004921110.

6. Clear-bottomed white 96-well microplates. CorningCostar Cat. No. 3903. Tel: 18004921110.

7. PCRE-luc-CHOK1 cell line generated by stable transfection of PCRE-luc plasmid (Stratagene, Catalog #219075) into CHOK1 cells (ATCC #CRL9618).

PREPARATION OF REAGENTS AND LUCIFERASE STANDARDS
The kit instructions were followed for preparation of the reagent.
1. PBS-BSA solution was prepared by adding 1% bovine serum albumin to Dulbeccos PBS 1X.

2. The firefly luciferase stock standard (1 mg/mL) was prepared in PBS-BSA. It was divided into 40 L aliquots and stored at -70C until needed.

3. For the dynamic range standard curve, the stock luciferase standard was diluted (10 L to 1000 L) in PBS-BSA, followed by 1-to-10 serial dilution in PBS-BSA. The diluted standards (10 g/mL to 10 pg/mL) were kept on ice until use.

4. For the low end standard curve, the stock luciferase standard was diluted (10 L to 1000 L) in PBS-BSA, followed by 2 additional 1-to100 serial dilutions. The final working standards were prepared by 1-to-50 dilution of the previous solution, followed by 1-to-2 serial dilutions. The diluted standards (0.625 to 20 pg/mL) were kept on ice until use.

5. The luciferase assay substrate (Component A) was reconstituted by adding 10 mL of luciferase assay buffer (Component B) to the vial, followed by gentle swirling and allowing it to stand at room temperature for approximately 1/2 hour. That substrate/buffer solution was then diluted 1:1 with PBS 1X. (Thus the final concentration was comparable to that intended in the kit instructions, where the reagent and cell suspension is mixed in a 1:1 ratio.)

PREPARATION OF THE LMAX
1. The M injector was cleaned by pumping 75% ethanol through the system and allowing it to stand for one hour, followed by rinsing generously with deionized water (5 washes, 99 cycles each).

2. The M injector was filled with the luciferase reagent by pumping 2 mL through it.

RECOMBINANT LUCIFERASE ASSAY PROCEDURE
1. Diluted luciferase standards and buffer blank (10 L each) were pipetted into microplate wells in quadruplicate. The final luciferase content ranged from 100 fg/well to 100 ng/well for the dynamic range standard curve and 6.25 to 200 fg/well for the low-end standard curve.

2. For the fast kinetic assays, the Lmax was set to inject 100 uL (injector M) and then to immediately begin integrating at consecutive 1-sec intervals.

3. For the endpoint assays, the Lmax was set up to inject 100 L/well, followed by a 2-second delay and a 1-second read.

PREPARATION OF CELLS AND ASSAY OF CELLULAR LUCIFERASE
1. PCRE-luc-CHOK1 cells were split into 96-well clear-bottom white microplates at 20,000 to 100,000 cells/well (100 L/well). Prior to use, they were treated with or without 1 mM cAMP for 4 hrs.

2. For the fast kinetic assays, the Lmax was set to inject 100 uL (injector M) and then to immediately begin integrating at consecutive 1-sec intervals.

3. For the endpoint assays, the Lmax was set up to inject 100 L/well, followed by a 2-second delay and a 1-second read.

REACTIONS PROFILES
The response of recombinant firefly luciferase to the addition of reagent is shown in Figure 2. The flash of light reached its maximum within 2 seconds and decayed thereafter.

The response of PCRE-luc-CHOK1 cells to the addition of reagent is shown in Figure 3. cAMP-treated cells (upper plot) had 5-fold increase in signal compared to the untreated controls (lower plot). The kinetic profiles were remarkably similar to that of recombinant luciferase: the light intensity was maximal within approximately 2 seconds and decayed thereafter to a much lower steady state. These results indicated that the CLIPR Luciferase Assay caused essentially instantaneous cell lysis.

DYNAMIC RANGE AND SENSITIVITY
Figure 4 illustrates typical results obtained with recombinant firefly luciferasestandards. The dynamic range spans at least 6 logs.

The low end of the standard curve is shown in Figure 5. The lower limit of detection (based on amount giving a signal 3 positive SD of the blank) is approximately 2 fg/well, and the lower limit of quantitation (based on amount giving a signal 10 positive SD of the blank) is approximately 6 fg/well.

PCRE-LUC-CHOK1 CELL RESPONSE AS A FUNCTION OF CELL NUMBER
The magnitude of the luminescent signal clearly increases as the number ofcAMP-treated cells is increased (Figure 6).

SUMMARY
Molecular Devices CLIPR Luciferase Assay Kit makes the measurements easy and sensitive with a lower limit of quantitation for (recombinant enzyme) of approximately 6 fg/well. For measuring luciferase in cells, lysis and enzyme activity occur almost instantaneously in a flash-type reaction. The Lmax microplate luminometer is an excellent instrument for measurement of luciferase activity as a reporter gene in a microplate format. In addition, SOFTmax PRO for Lmax provides a powerful and convenient instrument control, data analysis and presentation package.

REFERENCES
1. DeLuca, M.A. and W.D. McElroy, (1978) in Meth. Enzymol. vol 53:, p.3

Reading with no delay may give slightly better results in view of the fact that both the flash reaction and cell lysis appear to be maximal within 2 seconds (see Figures 2 & 3).

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