Reporter assay

Cells are loaded with a Ca -sensitive fluorescent dye (Fluo-3 AM, Ca-Green AM). 

Ex 488nm After stimulation with an agonist and excitation at 488 nm, the emission at 530 nm is increased. 

An antagonist is added before the agonist and Em 530nrn inhibits the effect of the agonist. 

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Cell based assays for NRs range from reporter gene assays to in vivo recruitment assays. The most reported of these is the GAL4 reporter assay. This assay takes advantage of the fact that the GAL4 response element of yeast does not exist in mammalian systems. 

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. 

Intracellular Enzymes Stem Cell Isolation Cellular Membrane Potential Mitochondrial Membrane Potential Gene Reporter Assay Gene Silencing (siRNA)... 

YES Yeast-based recombinant estrogen receptor-reporter assay... 

An example of a TIE approach is that described by Desbrow et al. [7]. In this work, the endocrine disrupting activity detected in effluents of seven UK WWTPs by means of a yeast-based screening assay [52] was mainly attributed to the presence of estradiol, estrone, and ethynylestradiol. However, to assess the estrogenic activity different bioassays may be used, e.g., the yeast-based recombinant estrogen receptor-reporter assay (YES), the MCF-7 cell proliferation (E-screen), and the estrogen receptor-mediated chemically activated... 

Microbes and metazoans are exposed to a variety of toxic stresses and have evolved appropriate defenses and repair mechanisms. Some of these systems are regulated at the protein level, but others are regulated at the transcriptional level, allowing the development of reporter assays. These transcriptional responses can be used to provide an earlier marker for genotoxin exposure in a whole population of cells, rather than the detection of the endpoints discussed above, in which genotoxic stress leads to fixation of mutations or chromosomal aberrations/damage in a small subpopulation. 

A positive result from a bacterial reporter assay would lower the ranking of the compound for development. The compound file should be aimotated with an alert for a confirmatory in vitro test (if the compound remains hve), in this case it might be a screening Ames at a later stage. There should also be a second alert to the lead optimization chemists. A third alert should still notify the genotoxicity safety assessment team that the compound is a priority for mechanistic investigation if the series continues, with the associated need for coordination of in vivo studies, this would be a lower priority than an Ames profiling positive. 

A positive result from a eukaryotic reporter assay would lower the ranking of a compound for development, alert lead optimization chemists and similarly trigger a confirmatory assay and alert safety assessment. 

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