Firefly luciferase assay

Brasier, A. R., Tate, J. E., and Habener, J. F. (1989) Optimized use of firefly luciferase assay as a reporter gene in mammalian tell lines. Biotechnology 7(10), 1116-1122. 

OTHER TECHNIQUES A number of other techniques have been used to determine specific organic phosphorus compounds in the environment, including a firefly luciferase assay for adenosine di- and triphosphate (Kaplan and Bott, 1985), a protein assay for cyclic adenosine monophosphate (Francko and Wetzel, 1982) and cetryltrimethylammonium bromide precipitation of dissolved RNA and DNA in marine and freshwaters (Karl and Bailiff, 1989). However, none of these are regularly used in studies of organic phosphorus dynamics in aquatic ecosystems. 

Bioluminescence in vitro chemosensitivity assays are now used to assess the sensitivity of tumor cells (obtained by surgical or needle biopsy) to different dmgs and combinations of dmgs. Cells are grown in microwell plates in the presence of the dmgs at various concentrations. If the tumor cells are sensitive to the dmg then they do not grow, hence total extracted cellular ATP, measured using the bioluminescence firefly luciferase reaction, is low. This method has been used to optimize therapy for different soHd tumors and for leukemias (306). 

Ca2+-sensitive photoproteins, 367 coelenterazine, 362 coelenterazine enol-sulfate, 364 coelenterazine luciferase, 363 Cypridina luciferase, 366 Cypridina luciferin, 365 dehydrocoelenterazine, 365 stabilized coelenterazine, 364 Asteroidea, 231 Astronestbes, 338 Atolla, 91,140, 334 ATP, 3-5,10-16, 23-29 Aracbnocampa luminescence, 26 assay of firefly luciferase, 11 firefly bioluminescence, 3-5, 10-16... 

Luciferase assay. In this technique, firefly luciferase is used to measure small amounts of adenosine triphosphate (ATP) in a bacterial culture, ATP levels being reduced by the inhibitory action of aminoglycoside antibiotics. This method may find more application in the future as more active and reliable luciferase preparations become available. 

Cells are lysed for Firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay system (Promega), following the manufacturer s instructions. We use a multimode microplate reader with automatic injectors (FLUOROstar Optima from BMG Labtech, OfFenburg, Germany) for luminescence measurements. 

For luciferin, a firefly luciferase cosubstrate, another method of retention has been evaluated which consisted of incorporating the substrate in acrylic microspheres during their formation, these last being then confined in a polymeric matrix31. Using the suitable co-immobilized enzymes (adenylate kinase and creatine kinase), the three adenylic nucleotides (ATP, ADP and AMP) could be assayed continuously and reproducibly with a selfcontainment working time of 3 h. 

Firefly luciferase, together with luciferases from other organisms, can be used as the labeling enzyme in immunoassays and nucleic acid assays [25], Recently, a highly sensitive BL ELISA using firefly luciferase was applied to thyreo-... 

In the method shown in Figure 9B, a firefly luciferase gene is introduced for sensitive bioluminescent detection of target DNA [5], The luciferase-coding DNA requires no posttranslational modification, and the activity of the luciferase produced can be readily measured in the transcription/translation mixture without prior purification. In this assay system, the digoxigenin-labeled probe is first immobilized to polystyrene wells coated with antidigoxigenin antibody. The target... 

Bioluminescence provides the basis for sensitive enzymic assay methods both for substrate assays and coupled enzyme assays. Firefly luciferase (EC 1.13.12.5) catalyses the production of light (540-600 nm) by the oxidation of luciferin (d-LH2) (Figure 8.18). 

Lundin, A., Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites, Methods Enzymol., 305, 346, 2000. 

An additional screening test for TCDD-like (aryl hydrocarbon receptor, AhR, active) chemicals has been developed (Garrison et al. 1996) and is available commercially (Anonymous 1997). Dubbed the CALUX (for chemically activated luciferase gene expression) system, the assay is based on recombinant cell lines into which researchers have inserted a firefly luciferase gene. When exposed to dioxin-like compounds, the recombinant cells luminesce. The method is sensitive to ppt levels of 2,3,7,8-TCDD equivalents in blood, serum, and milk (Anonymous 1997). Samples testing positive can be subjected to more definitive and specific analytical testing. 

Living cells generate adenosine triphosphate (ATP) that can readily be detected by enzyme assays, e.g. luciferin emits light when exposed to firefly luciferase in the presence of ATP light... 

Glow luminescence techniques have been used extensively with luciferases as reporter genes in cell-based assays. An overview of such assays is given in Section 10.3.2 Reporter Assays below. Luciferases are enzymes which catalyze bio-luminescent reactions. Two forms are used as reporters, one originating from the firefly (firefly luciferase) and the other from Rmilla (Renilla luciferase). Due to their different origins, the enzyme structures and their respective substrates are quite different While Rmilla luciferase catalyzes the oxidation of coelenterazine, the substrate of firefly luciferase is the beetle luciferin, which is oxidized in the presence of ATP and Mg as depicted in Fig. 17. 

---