title: Real-time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no-wash, no-lyse kinetic method.
abstract: Nitric oxide (NO) and its related reactive nitrogen species (RNS) and reactive oxygen species (ROS) are crucial in monocyte responses against pathogens and also in inflammatory conditions. Central to both processes is the generation of the strong oxidant peroxynitrite (ONOO) by a fast reaction between NO and superoxide anion. ONOO is a biochemical junction for ROS- and RNS cytotoxicity and causes protein nitrosylation. Circulating by-products of protein nitrosylation are early biomarkers of inflammation-based conditions, including minimal hepatic encephalopathy in cirrhotic patients (Montoliu et al., Am J Gastroenterol 2011; 106:1629-1637). In this context, we have designed a novel no-wash, no-lyse real-time flow cytometry assay to detect and follow-up the NO- and superoxide-driven generation of ONOO in peripheral blood monocytes.
  
  
  
  Whole blood samples were stained with CD45 and CD14 antibodies plus one of a series of fluorescent probes sensitive to RNS, ROS, or glutathione, namely 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, dihydrorhodamine 123, MitoSOX Red, dihydroethidium, and 5-chloromethylfluorescein diacetate. Samples were exposed sequentially to a NO donor and three different superoxide donors, and analyzed in real time by kinetic flow cytometry. Relevant kinetic descriptors, such as the rate of fluorescence change, were calculated from the kinetic plot.
  
  
  
  The generation of ONOO, which consumes both NO and superoxide, led to a decrease in the intensity of the cellular fluorescence of the probes sensitive to these molecules.
  
  
  
  This is a fast and simple assay that may be used to monitor the intracellular generation of ONOO in physiological, pathological, and pharmacological contexts. © 2015 International Clinical Cytometry Society.
authors.affiliation: Laboratory of Cytomics, Mix Research Unit, Universidad De Valencia and Centro De Investigacion Principe Felipe, Valencia, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Balaguer
authors.affiliation: Laboratory of Cytomics, Mix Research Unit, Universidad De Valencia and Centro De Investigacion Principe Felipe, Valencia, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Diaz
authors.affiliation: Laboratory of Cytomics, Mix Research Unit, Universidad De Valencia and Centro De Investigacion Principe Felipe, Valencia, Spain.
authors.forename: Angela
authors.initials: A
authors.lastname: Gomes
authors.affiliation: Fundacion Investigacion Hospital Clınico Universitario De Valencia, INCLIVA, Valencia, Spain.
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Herrera
authors.affiliation: Laboratory of Cytomics, Mix Research Unit, Universidad De Valencia and Centro De Investigacion Principe Felipe, Valencia, Spain.
authors.forename: José-Enrique
authors.initials: JE
authors.lastname: O'Connor
authors.affiliation: Fundacion Investigacion Hospital Clınico Universitario De Valencia, INCLIVA, Valencia, Spain.
authors.forename: Amparo
authors.initials: A
authors.lastname: Urios
authors.affiliation: Laboratory of Neurobiology, Centro De Investigación Principe Felipe, Valencia, Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Felipo
authors.affiliation: Fundacion Investigacion Hospital Clınico Universitario De Valencia, INCLIVA, Valencia, Spain.
authors.forename: Carmina
authors.initials: C
authors.lastname: Montoliu
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007249:Inflammation / Q000097:blood / Q000473:pathology; D007700:Kinetics; D017493:Leukocyte Common Antigens / Q000097:blood*; D018950:Lipopolysaccharide Receptors / Q000097:blood*; D009000:Monocytes / Q000378:metabolism; D009569:Nitric Oxide / Q000097:blood*; D030421:Peroxynitrous Acid / Q000097:blood / Q000276:immunology / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000097:blood; D017382:Reactive Oxygen Species / Q000097:blood; D013481:Superoxides / Q000097:blood*
chemical_list: D005456:Fluorescent Dyes; D018950:Lipopolysaccharide Receptors; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
keywords: monocyte; nitrosative stress; oxidative stress; protein nitrosylation; real-time cytometry