Development of a High-Throughput Cell-Based Assay for Superoxide Production in HL-60 Cells

• Published in: SLAS discovery 2010-04, Vol.15 (4), p.388-397
• Main Authors: Seitz, Patricia M., Cooper, Rona, Gatto, Jr, Gregory J., Ramon, Fernando, Sweitzer, Thomas D., Johns, Douglas G., Davenport, Elizabeth A., Ames, Robert S., Kallal, Lorena A.
• Format: Article
• Language: English
• Subjects: frozen cells; high-throughput screening; HL-60 cells; Oxyburst Green BSA; superoxide
• ISSN: 2472-5552; 2472-5560
• DOI: 10.1177/1087057109359687

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC50 reproducibility and met a desired Z′ value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 µM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.