Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase.
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 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 microM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 +/- 6% and Nox4 siRNA 83 +/- 7% of control) and lucigenin chemiluminescence (Nox2; 54 +/- 6% and Nox4 74 +/- 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.
Peshavariya
Hitesh M
HM
Cytoprotection Pharmacology Laboratory, Bernard O'Brien Institute of Microsurgery, University of Melbourne, Melbourne, Vic., Australia.
Dusting
Gregory James
GJ
Selemidis
Stavros
S
D000166:Acridines / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004996:Ethidium / Q000031:analogs & derivatives* / Q000737:chemistry; D005453:Fluorescence; D018922:HL-60 Cells; D006801:Humans; D049449:Luminescence; D049408:Luminescent Agents / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*; D019820:Xanthine / Q000494:pharmacology; D014969:Xanthine Oxidase / Q000378:metabolism
D000166:Acridines; D049408:Luminescent Agents; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; C067883:dihydroethidium; D013481:Superoxides; D019820:Xanthine; C033472:10,10'-dimethyl-9,9'-biacridinium; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D004996:Ethidium