Protocol

Cell-Based Assays for High-Throughput Screening

Volume 486 of the series Methods in Molecular Biology pp 151-165

Date:

High-Content Screening: Flow Cytometry Analysis

  • Bruce S. EdwardsAffiliated withCancer Research and Treatment Center, University of New Mexico Email author 
  • , Susan M. YoungAffiliated withCancer Research and Treatment Center, University of New Mexico
  • , Irena Ivnitsky-SteeleAffiliated withCancer Research and Treatment Center, University of New Mexico
  • , Richard D. YeAffiliated withCancer Research and Treatment Center, University of New Mexico
  • , Eric R. ProssnitzAffiliated withCancer Research and Treatment Center, University of New Mexico
  • , Larry A. SklarAffiliated withCancer Research and Treatment Center, University of New Mexico

* Final gross prices may vary according to local VAT.

Get Access

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.

Key words

Flow cytometry Fluorescence Formylpeptide receptor Formylpeptide receptor-like 1 High-content screening High-throughput screening NIH Molecular Libraries Screening Center Network