High-Content Screening: Flow Cytometry Analysis
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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.