A semi-automated micro-assay for H2O2 release by human blood monocytes and mouse peritoneal macrophages

Abstract

H2O2 secreted by mononuclear phagocytes can be detected by monitoring the horseradish peroxidase-catalyzed oxidation of fluorescent scopoletin. This technique has been adapted to a semi-automated micro-scale with the aid of automatic fluorescence and absorbance micro-culture plate readers to measure H2O2 and protein, respectively, in the same culture wells. With these adaptations the assay can accurately and precisely detect as little as 0.1 nmol H2O2 or 1 microgram cell protein, permitting the calculation of specific secretion (nmol H2O2/mg cell protein) from as few as 2 X 10(4) human blood monocytes or mouse peritoneal macrophages. Cumulative H2O2 secretion in individual wells may be recorded non-destructively at frequent intervals for time course measurements. Less than 1 min is required to record the fluorescence in all 96 wells of a micro-culture plate. The assay is highly reproducible, with standard deviations for triplicates typically less than 5-10% of the mean, and gives values in close agreement with those obtained in 10-fold larger samples by previous methods. Using this assay it is feasible to process 1000 samples per day, with order of magnitude savings in labor, cells, sera, media, cytokines, and reagents compared to earlier forms of the assay. The assay is useful in evaluating the cellular effects of cytokines and for assaying their activity in chromatographic fractions and hybridoma cultures. We are currently using the assay to monitor the administration of interferon-gamma to patients with neoplasia.