Development of a high-throughput cell-based assay for superoxide production in HL-60 cells.
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 IC(50) reproducibility and met a desired Z' value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 microM 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.
Seitz
Patricia M
PM
Department of Screening and Compound Profiling, GlaxoSmithKline R&D, Collegeville, Pennsylvania 19426, USA.
Cooper
Rona
R
Gatto
Gregory J
GJ
Ramon
Fernando
F
Sweitzer
Thomas D
TD
Johns
Douglas G
DG
Davenport
Elizabeth A
EA
Ames
Robert S
RS
Kallal
Lorena A
LA
D045304:Cytochromes c / Q000378:metabolism; D018922:HL-60 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009861:Onium Compounds / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D009861:Onium Compounds; D013481:Superoxides; C007517:diphenyleneiodonium; D045304:Cytochromes c; D013755:Tetradecanoylphorbol Acetate