Summary
The HyperCyt® high-throughput (HT) flow cytometry sampling platform uses a peristaltic pump, in combination with an autosampler, and a novel approach to data collection, to circumvent time-delay bottlenecks of conventional flow cytometry. This approach also dramatically reduces the amount of sample aspirated for each analysis, typically requiring ~2 μL per sample while making quantitative fluorescence measurements of 40 or more samples per minute with thousands to tens of thousands of cells in each sample. Here, we describe a simple robust screening assay that exploits the high-content measurement capabilities of the flow cytometer to simultaneously probe the binding of test compounds to two different receptors in a common assay volume, a duplex assay format. The ability of the flow cytometer to distinguish cell-bound from free fluorophore is also exploited to eliminate wash steps during assay setup. HT flow cytometry with this assay has allowed efficient screening of tens of thousands of small molecules from the NIH Small-Molecule Repository to identify selective ligands for two related G-protein-coupled receptors, the formylpeptide receptor and formylpeptide receptor-like 1.
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Acknowledgments
This work was supported by NIH R03 MH076381-01, U54 MH074425-01, the New Mexico Molecular Libraries Screening Center, the University of New Mexico Shared Flow Cytometry Resource, and Cancer Research and Treatment Center.
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Edwards, B.S., Young, S.M., Ivnitsky-Steele, I., Ye, R.D., Prossnitz, E.R., Sklar, L.A. (2009). High-Content Screening: Flow Cytometry Analysis. In: Clemons, P., Tolliday, N., Wagner, B. (eds) Cell-Based Assays for High-Throughput Screening. Methods in Molecular Biology, vol 486. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-545-3_11
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DOI: https://doi.org/10.1007/978-1-60327-545-3_11
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