Flow cytometry study of polymorphonuclear neutrophil oxidative burst: a comparison of three fluorescent probes

Background: The use of 2',7'-dichlorofluorescein diacetate (DCFH), dihydrorhodamine 123 (DHR) and hydroethidine (HE) has been described for detecting respiratory burst activity by flow cytometry in polymorphonuclear neutrophil (PMN) suspension. However, their specificities for reactive oxygen species are not well defined. We investigated the reactivity of these probes for detecting superoxide anion (O-2(.-)), hydrogen peroxide (H2O2) and/or nitric oxide (NO.)-dependent mechanisms. Methods: PMNs (10(6)/ml) were preincubated for 15 min at 37 degreesC with DCFH (5 mumol/l), DHR (1 mumol/l) or HE (10 mumol/l). Cell suspensions were then split for each probe into five different aliquots containing either no effector or one effector: N-ethylmaleimide (NEM, 150 mumol/l, NADPH oxidase inhibitor), sodium azide (NaN3, 50 mumol/l, peroxidase and catalase inhibitor), N-nitro-L-arginine methyl ester (L-NAME, 1.5 mumol/l, NO. synthase inhibitor) or H2O2 (30%). At the same time, PMNs were stimulated with phorbol myristate acetate (PMA, 10 mumol/l) for 10 min at 37 degreesC. Analyses were carried out on a Beckman-Coulter Epics XL equipped with an argon laser (488 nm). Green fluorescences from DCFH and DHR were measured in the FL1 channel and HE fluorescence was analyzed in the FL2 channel. Results: NaN3 decreased the fluorescence of PMNs incubated with DCFH, indicating that it needs a peroxidase activity to react with H2O2. L-NAME reduced the oxidation of DCFH, showing that it reacts with reactive nitrogen species. DHR was specifically responsive to H2O2 accumulation. HE seemed to be preferentially oxidized by O-2(.-). Conclusions: Hence the choice of the probe to be used depends on the reactive species of interest. (C) 2003 Elsevier Science B.V. All rights reserved.