A highly sensitive fluorescent micro-assay of H 2O 2 release from activated human leukocytes using a dihydroxyphenoxazine derivative

Abstract

This study describes a simple, reliable, highly sensitive and quantitative fluorescence microplate-assay of H 2O 2 from activated leukocytes using a novel horse radish peroxidase (HRP) substrate N-acetyl-3,7-dihydroxyphenoxazine (A6550). Unlike the widely used fluorescent HRP substrate scopoletin, A6550 is non-fluorescent and becomes highly fluorescent upon HRP-catalyzed H 2O 2 oxidation. Using 50 μM A6550, the change in fluorescence due to H 2O 2 generated from phorbol 12-myristate 13-acetate-activated human eosinophils and neutrophils is found to have a linear cell dose response up to 1.5×10 4 and 5×10 4 cells, respectively. The increase in fluorescence from A6550 is specifically due to H 2O 2 generation since it is inhibitable by catalase. Oxidized A6550 is found to be highly stable and the H 2O 2 dose response is linear as long as the ratio of A6550:H 2O 2 in the reaction mixture is higher than five. Unlike scopoletin, A6550 has a very low background, which changes little with time. In addition, the high fluorescent yield of oxidized A6550 results in an increased sensitivity for the detection of H 2O 2. When the concentrations of A6550 and HRP were 10 μM and 0.2 U/ml, respectively, as low as 2 pmol of H 2O 2 could be reliably measured. The sensitivity of A6550/H 2O 2 assay is found to be at least 10-fold higher than with scopoletin as the HRP substrate. The protocol described in this study using A6550 to measure H 2O 2 release from activated granulocytes can be easily adapted to other cell types which generate H 2O 2.