High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands.
High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (approximately 2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [K(i)]=14 microM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.
Young
Susan M
SM
Cytometry and Department of Pathology, Cancer Research and Treatment Center, University of New Mexico, Health Sciences Center, Albuquerque, NM 87131, USA.
Bologa
Cristian
C
Prossnitz
Eric R
ER
Oprea
Tudor I
TI
Sklar
Larry A
LA
Edwards
Bruce S
BS
D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008024:Ligands; D044042:Receptors, Formyl Peptide / Q000378:metabolism*; D012680:Sensitivity and Specificity; D020298:U937 Cells
D008024:Ligands; D044042:Receptors, Formyl Peptide