A Saccharomyces cerevisiae cell-based quantitative beta-galactosidase assay compatible with robotic handling and high-throughput screening.
ABSTRACT Reporter-gene assays that employ the Escherichia coli lacZ gene are ubiquitously employed in biological research. However, we were not able to readily identify a quantitative method that worked reliably with yeast (Saccharomyces cerevisiae) cells and that was compatible with high-throughput screening and robotic liquid handling tools. We have therefore adapted a commercially available assay employing a 6-O-beta-galactopyranosyl-luciferin substrate to provide the required sensitivity with minimal sample handling times. Our assay uses only one-tenth of the reagents suggested by the reagent manufacturer (Promega) for equivalent assays with mammalian cell cultures and produces rapid, sensitive and reproducible analysis with as little as 1 microl yeast cell culture and with < 100 cells. We demonstrate that the assay is compatible with yeast strains generated by the systematic yeast deletion project and functions equally well with genomically integrated or plasmid-encoded lacZ reporters and with cells grown in complex or defined media. The high-sensitivity, miniaturized format reduced sample handling required will make this assay useful for a wide range of applications.
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ABSTRACT: Developing high-throughput assays to screen marine extracts for bioactive compounds presents both conceptual and technical challenges. One major challenge is to develop assays that have well-grounded ecological and evolutionary rationales. In this review we propose that a specific group of ligand-activated transcription factors are particularly well-suited to act as sensors in such bioassays. More specifically, xenobiotic-activated nuclear receptors (XANRs) regulate transcription of genes involved in xenobiotic detoxification. XANR ligand-binding domains (LBDs) may adaptively evolve to bind those bioactive, and potentially toxic, compounds to which organisms are normally exposed to through their specific diets. A brief overview of the function and taxonomic distribution of both vertebrate and invertebrate XANRs is first provided. Proof-of-concept experiments are then described which confirm that a filter-feeding marine invertebrate XANR LBD is activated by marine bioactive compounds. We speculate that increasing access to marine invertebrate genome sequence data, in combination with the expression of functional recombinant marine invertebrate XANR LBDs, will facilitate the generation of high-throughput bioassays/biosensors of widely differing specificities, but all based on activation of XANR LBDs. Such assays may find application in screening marine extracts for bioactive compounds that could act as drug lead compounds.Marine Drugs 11/2014; 12(11):5590-5618. DOI:10.3390/md12115590 · 3.51 Impact Factor
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ABSTRACT: Parallel high-throughput automated assays are described for the measurement of cell growth and β-galactosidase reporter gene expression from a single culture of the yeast S. cerevisiae. The dual assay measures the effect of test compounds on expression of a specific gene of interest linked to the β-galactosidase reporter gene, and simultaneously tests for compound toxicity and other effects on cell growth. Examples of assay development and validation results are used to illustrate how this protocol may be used to screen two yeast cell lines in parallel. Yeast cells are grown overnight in V-bottom polypropylene 384-well plates, after which portions of the cell suspension are transferred to clear and to white flat-bottom 384-well plates for measurement of cell growth and reporter gene expression, respectively. Cell growth is determined by measurement of absorbance at 595 nm, and β-galactosidase expression is quantified by Beta-Glo, a commercially available luminescent β-galactosidase substrate. Curr. Protoc. Chem. Biol. 3:1-14 © 2011 by John Wiley & Sons, Inc.03/2011; 3(1):1-14. DOI:10.1002/9780470559277.ch100119
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ABSTRACT: Background aims. Current methods of mesenchymal stromal cell (MSC) cryopreservation result in variable post-thaw recovery and phenotypic changes caused by freezing. The objective of this investigation was to determine the influence of ex vivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing. Methods. Human bone marrow aspirate was used. MSCs were isolated and cells were assessed for total count, viability, apoptosis and senescence over 6 passages (8-10 doublings/passage) in ex vivo culture. One half of cells harvested at each passage were replated for continued culture and the other half were frozen at 1 degrees C/min in a controlled-rate freezer. Frozen samples were stored in liquid nitrogen, thawed and reassessed for total cell count, viability and senescence immediately and 48 h after thaw. Results. Viability did not differ significantly between samples before freeze or after thaw. Senescence increased over time in pre-freeze culture and was significantly higher in one sample that had growth arrest both before freeze and after thaw. Freezing resulted in similar initial post-thaw recovery in all samples, but 48-h post-thaw growth arrest was observed in the sample with high senescence only. Conclusions. High pre-freeze senescence appears to correlate with poor post-thaw function in MSC samples, but additional studies are necessary to obtain a sample sizes large enough to quantify results.Cytotherapy 10/2014; 17(1). DOI:10.1016/j.jcyt.2014.06.008 · 3.10 Impact Factor