Edwards Bruce S, Young Susan M, Ivnitsky-Steele Irena, Ye Richard D, Prossnitz Eric R, Sklar Larry A
Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM, USA.
Methods Mol Biol. 2009;486:151-65. doi: 10.1007/978-1-60327-545-3_11.
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 microL 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 simicroltaneously 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.
HyperCyt高通量（HT）流式细胞术采样平台使用蠕动泵，结合自动进样器和一种新颖的数据收集方法，以规避传统流式细胞术的时间延迟瓶颈。这种方法还显著减少了每次分析所吸取的样品量，通常每个样品需要约2微升，同时每分钟对40个或更多样品进行定量荧光测量，每个样品中有数千到数万个细胞。在此，我们描述了一种简单可靠的筛选测定法，该方法利用流式细胞仪的高内涵测量能力，在一个共同的测定体积中同时探测测试化合物与两种不同受体的结合，即双工测定形式。流式细胞仪区分细胞结合荧光团和游离荧光团的能力也被用于在测定设置过程中消除洗涤步骤。使用这种测定法的HT流式细胞术能够有效地从美国国立卫生研究院小分子文库中筛选数万个小分子，以鉴定两种相关G蛋白偶联受体（甲酰肽受体和甲酰肽受体样1）的选择性配体。
