Is the neutrophil reactive oxygen species production measured by luminol and lucigenin chemiluminescence intra or extracellular? Comparison with DCFH-DA flow cytometry and cytochrome c reduction.
BACKGROUND
Polymorphonuclear neutrophils (PMNs) are crucial in host defense against invading microorganisms through reactive oxygen species (ROS) production. However, generated ROS released in excess into media can damage the host tissue. It is therefore essential, when exploring oxygen species production, to discriminate between its intracellular (IC) and extracellular (EC) localization. Several methods of ROS detection are commonly used. However, the literature shows that it is not always clear whether the species detected are IC or EC, especially with the chemiluminescence technique.


METHODS
We compared PMN ROS production, determined by chemiluminescence, using two different probes (luminol and lucigenin) with that measured by 2'-7'-dichlorofluorescin diacetate (DCFH-DA) flow cytometry for IC production and by cytochrome c reduction for EC production.


RESULTS
We found that luminol-dependent chemiluminescence explored IC ROS production more specifically (r=0.77, p<0.01: correlation between luminol-amplified chemiluminescence and DCFH-DA flow cytometry). Lucigenin-amplified chemiluminescence and cytochrome c reduction were closely related (r=0.55, p<0.01).


CONCLUSIONS
Luminometry detection can thus afford reproducible information on intracellular ROS kinetic production using luminol and extracellular ROS detection using lucigenin, simply and at low cost.
Caldefie-Chézet
Florence
F
Laboratoire de Biochimie, Biologie Moléculaire et Nutrition, EA 2416, Centre de Recherche en Nutrition Humaine, Faculté de Pharmacie, 28 place Henri-Dunant. B.P. 38, 63001 Clermont-Ferrand Cedex 1, France. F.Caldefie-Chezet@u-clermont1.fr
Walrand
Stephane
S
Moinard
Christophe
C
Tridon
Arlette
A
Chassagne
Jacques
J
Vasson
Marie-Paule
MP
D000166:Acridines / Q000378:metabolism*; D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D005434:Flow Cytometry; D005452:Fluoresceins; D007424:Intracellular Fluid / Q000378:metabolism*; D007700:Kinetics; D008163:Luminescent Measurements; D008165:Luminol / Q000378:metabolism*; D008297:Male; D009504:Neutrophils / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000166:Acridines; D003574:Cytochrome c Group; D005452:Fluoresceins; D017382:Reactive Oxygen Species; C029569:diacetyldichlorofluorescein; C033472:10,10'-dimethyl-9,9'-biacridinium; D008165:Luminol