Development of a High-Throughput Cell-Based Assay for Superoxide Production in HL-60 Cells
| Autor: | Thomas D. Sweitzer, Gatto Gregory J, Elizabeth A. Davenport, Douglas G. Johns, Robert S. Ames, Patricia M. Seitz, Rona Cooper, Fernando Ramón, Lorena A. Kallal |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Time Factors
High-throughput screening Cell HL-60 Cells Biochemistry Cryopreservation Analytical Chemistry chemistry.chemical_compound Onium Compounds Superoxides medicine Extracellular Humans Lucigenin Chemistry Superoxide Cytochromes c Fluorescence Molecular biology High-Throughput Screening Assays Kinetics medicine.anatomical_structure Reagent Tetradecanoylphorbol Acetate Molecular Medicine Biotechnology |
| Zdroj: | SLAS Discovery. 15:388-397 |
| ISSN: | 2472-5552 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|