Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase

Abstract

All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 gen..