title: 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.
abstract: 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.
authors.affiliation: 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
authors.forename: Florence
authors.initials: F
authors.lastname: Caldefie-Chézet
authors.forename: Stephane
authors.initials: S
authors.lastname: Walrand
authors.forename: Christophe
authors.initials: C
authors.lastname: Moinard
authors.forename: Arlette
authors.initials: A
authors.lastname: Tridon
authors.forename: Jacques
authors.initials: J
authors.lastname: Chassagne
authors.forename: Marie-Paule
authors.initials: MP
authors.lastname: Vasson
mesh_terms: 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
chemical_list: D000166:Acridines; D003574:Cytochrome c Group; D005452:Fluoresceins; D017382:Reactive Oxygen Species; C029569:diacetyldichlorofluorescein; C033472:10,10'-dimethyl-9,9'-biacridinium; D008165:Luminol