High Content Screening: Flow Cytometry Analysis #MMPMID19347622
Edwards BS; Young SM; Ivnitsky-Steele I; Ye RD; Prossnitz ER; Sklar LA
Methods Mol Biol 2009[]; 486 (ä): 151-65 PMID19347622show ga
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 ?l per sample while making quantitative fluorescence measurements of 40 or more samples per min 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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Methods+Mol+Biol 2009 ; 486 (ä): 151-65
