Real-time cytometric assay of nitric oxide and superoxide interaction in peripheral blood monocytes: A no-wash, no-lyse kinetic method

Autores de CIPF

• Jose Enrique O'connor Blasco

  Autor

• 

  Amparo Urios Lluch

  Autor

• 

  Vicente Felipo Orts

  Autor

• Carmina Montoliu Felix

  Autor

Participantes ajenos a CIPF

• Balaguer, S
• Diaz, L
• Gomes, A
• Herrera, G

Grupos de Investigación

• Laboratorio de Neurobiología

  

  Jefe/a de Grupo

  Vicente Felipo Orts

• Unidad Mixta CIPF-INCLIVA de Deterioro Neurológico

• Unidad Mixta CIPF-UV de Citómica

Abstract

BackgroundNitric 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. MethodsWhole 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. ResultsThe 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. ConclusionThis is a fast and simple assay that may be used to monitor the intracellular generation of ONOO in physiological, pathological, and pharmacological contexts. (c) 2015 International Clinical Cytometry Society

Keywords

• oxidative stress; nitrosative stress; monocyte; real-time cytometry; protein nitrosylation