Abstract
A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.
Keywords: Cell-based assay, endocrine-disrupting compounds, high throughput assay, luciferase, nuclear receptors, saccharomyces cerevisiae, REACH, high throughput screening, Biomek NXP, Victor3 1420 Multilabel Counter, GraphPad Prism 4, BMAER, BMAAREluc/AR, BMAAhR/ ARNT, estrogen receptor, ER yeast assay, D-luciferin, AR assay, LOD, 17-estradiol, Dihydro- testosterone, Bentzo-a-pyrene, AhR assays, Z' factor
Combinatorial Chemistry & High Throughput Screening
Title: Miniaturization of a Panel of High Throughput Yeast-Cell-Based Nuclear Receptor Assays in 384- and 1536-Well Microplates
Volume: 14 Issue: 1
Author(s): Johanna Rajasarkka and Marko Virta
Affiliation:
Keywords: Cell-based assay, endocrine-disrupting compounds, high throughput assay, luciferase, nuclear receptors, saccharomyces cerevisiae, REACH, high throughput screening, Biomek NXP, Victor3 1420 Multilabel Counter, GraphPad Prism 4, BMAER, BMAAREluc/AR, BMAAhR/ ARNT, estrogen receptor, ER yeast assay, D-luciferin, AR assay, LOD, 17-estradiol, Dihydro- testosterone, Bentzo-a-pyrene, AhR assays, Z' factor
Abstract: A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.
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Cite this article as:
Rajasarkka Johanna and Virta Marko, Miniaturization of a Panel of High Throughput Yeast-Cell-Based Nuclear Receptor Assays in 384- and 1536-Well Microplates, Combinatorial Chemistry & High Throughput Screening 2011; 14 (1) . https://dx.doi.org/10.2174/1386207311107010047
DOI https://dx.doi.org/10.2174/1386207311107010047 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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