High-content screening: flow cytometry analysis.

Area of Science:

• Biochemistry
• Pharmacology
• Analytical Chemistry

Background:

• Conventional flow cytometry faces time-delay bottlenecks and requires large sample volumes.
• High-throughput screening (HTS) is crucial for drug discovery and identifying receptor ligands.
• G-protein-coupled receptors (GPCRs) are important drug targets, necessitating efficient screening methods.

Purpose of the Study:

• To develop a robust, high-throughput flow cytometry assay for screening small molecules.
• To simultaneously probe the binding of compounds to two distinct receptors in a duplex assay format.
• To identify selective ligands for formylpeptide receptor (FPR) and formylpeptide receptor-like 1 (FPRL1).

Main Methods:

• Utilized the HyperCyt high-throughput (HT) flow cytometry platform with a peristaltic pump and autosampler.
• Developed a duplex assay to simultaneously measure compound binding to two receptors in a single sample volume.
• Leveraged the flow cytometer's ability to differentiate cell-bound from free fluorophores, eliminating wash steps.

Main Results:

• Screened tens of thousands of small molecules from the NIH Small-Molecule Repository.
• Achieved quantitative fluorescence measurements of 40+ samples per minute with minimal sample volume (~2 microL).
• Successfully identified selective ligands for FPR and FPRL1.

Conclusions:

• HT flow cytometry, coupled with the developed duplex assay, offers an efficient method for screening small molecules.
• This approach significantly reduces sample consumption and assay time, overcoming limitations of conventional flow cytometry.
• The assay is effective for identifying selective ligands for GPCRs, facilitating drug discovery efforts.