title: Adaptation and validation of DNA synthesis detection by fluorescent dye derivatization for high-throughput screening.
abstract: Cellular proliferation is fundamental to organism development, tissue renewal, and diverse disease states such as cancer. In vitro measurement of proliferation by high-throughput screening allows rapid characterization of the effects of small-molecule or genetic treatments on primary and established cell lines. Current assays that directly measure the cell cycle are not amenable to high-throughput processing and analysis. Here we report the adaptation of the chemical method for detecting DNA synthesis by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into both high-throughput liquid handling and high-content imaging analysis. We demonstrate that chemical detection of EdU incorporation is effective for high-resolution analysis and quantitation of DNA synthesis by high-content imaging. To validate this assay platform we used treatments of MCF10A cells with media supplements and pharmacological inhibitors that are known to affect cell proliferation. Treatments with specific kinase inhibitors indicate that EGF and serum stimulation employs both the mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K)/AKT signaling networks. As described here, this method is fast, reliable, and inexpensive and yields robust data that can be easily interpreted.
authors.affiliation: University of Queensland Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital, Brisbane, Australia.
authors.forename: Max V
authors.initials: MV
authors.lastname: Ranall
authors.forename: Brian G
authors.initials: BG
authors.lastname: Gabrielli
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Gonda
mesh_terms: D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000096:biosynthesis*; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007211:Indoles; D008856:Microscopy, Fluorescence; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results
chemical_list: D005456:Fluorescent Dyes; D007211:Indoles; D047428:Protein Kinase Inhibitors; C007293:DAPI; D004815:Epidermal Growth Factor; D004247:DNA; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine