Author(s): Peshavariya HM, Dusting GJ, Selemidis S
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.
Referred From: https://www.ncbi.nlm.nih.gov/pubmed/17516243
Author(s): Bast A, Haenen GR
Author(s): Biesalski HK, Grune T, Tinz J, Zöllner I, Blumberg JB
Author(s): Weseler AR, Bast A
Author(s): He HP, Corke H
Author(s): Tinkel J, Hassanain H, Khouri SJ
Author(s): Calabrese V, Cornelius C, Dinkova-Kostova AT, Iavicoli I, Di Paola R, et al.
Author(s): Birringer M
Author(s): Forman HJ, Davies KJ, Ursini F
Author(s): Yelisyeyeva O, Semen K, Zarkovic N, Kaminskyy D, Lutsyk O, et al.
Author(s): Tikekar RV, Ludescher RD, Karwe MV
Author(s): Medina-Franco JL, Giulianotti MA, Welmaker GS, Houghten RA
Author(s): Huerta-Ocampo JA, Barba de la Rosa AP
Author(s): Yelisyeyeva OP, Kamisnkyy DV, Cherkas AP, Ambarova LI, Vyshemyrska OR, et al.
Author(s): Yelisyeyeva OP, Semen KO, Ostrovska GV, Kaminskyy DV, Sirota TV, et al.
Author(s): Yelisyeyeva O, Cherkas A, Semen K, Kaminskyy D, Lutsyk A
Author(s): Sen CK, Khanna S, Roy S
Author(s): Floyd RA, Wiseman BB
Author(s): Sirota TV
Author(s): Wang H, Joseph JA
Author(s): Ruijters EJ, Weseler AR, Kicken C, Haenen GR, Bast A
Author(s): Pasko P, Bartn H, Zagrodizki P, Chlopicka J, Izewska A, et al.
Author(s): Martirosyan DM, Miroshnichenko LA, Kulakova SN, Pogojeva AV, Zoloedov VI
Author(s): Aoun M, Feillet-Coudray C, Fouret G, Chabi B, Crouzier D, et al.
Author(s): Chatgilialoglu C, Ferreri C
Author(s): Puca AA, Andrew P, Novelli V, Anselmi CV, Somalvico F, et al.
Author(s): Reynolds CM, Loscher CE, Moloney AP, Roche HM
Author(s): Warleta F, Campos M, Allouche Y, Sánchez-Quesada C, Ruiz-Mora J, et al.
Author(s): Misra HP, Fridovich I
Author(s): Qi S, den Hartog GJ, Bast A
Author(s): Bast A, Weseler AR, Haenen GR, den Hartog GJ