Balaguer Susana, Diaz Laura, Gomes Angela, Herrera Guadalupe, O'Connor José-Enrique, Urios Amparo, Felipo Vicente, Montoliu Carmina
Laboratory of Cytomics, Mix Research Unit, Universidad De Valencia and Centro De Investigacion Principe Felipe, Valencia, Spain.
Fundacion Investigacion Hospital Clınico Universitario De Valencia, INCLIVA, Valencia, Spain.
Cytometry B Clin Cytom. 2017 May;92(3):211-217. doi: 10.1002/cyto.b.21237. Epub 2015 May 27.
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.
一氧化氮（NO）及其相关的活性氮物质（RNS）和活性氧物质（ROS）在单核细胞对抗病原体的反应以及炎症状态中起着关键作用。这两个过程的核心是NO与超氧阴离子通过快速反应生成强氧化剂过氧亚硝酸盐（ONOO）。ONOO是ROS和RNS细胞毒性的生化连接点，并导致蛋白质亚硝基化。蛋白质亚硝基化的循环副产物是基于炎症的疾病的早期生物标志物，包括肝硬化患者的轻微肝性脑病（Montoliu等人，《美国胃肠病学杂志》2011年；106:1629 - 1637）。在此背景下，我们设计了一种新型的无需洗涤、无需裂解的实时流式细胞术检测方法，以检测和跟踪外周血单核细胞中由NO和超氧阴离子驱动的ONOO生成。
全血样本用CD45和CD14抗体以及一系列对RNS、ROS或谷胱甘肽敏感的荧光探针之一进行染色，即4 - 氨基 - 5 - 甲基氨基 - 2',7'-二氟荧光素二乙酸酯、二氢罗丹明123、MitoSOX Red、二氢乙锭和5 - 氯甲基荧光素二乙酸酯。样本依次暴露于NO供体和三种不同的超氧供体，并通过动力学流式细胞术进行实时分析。从动力学图中计算出相关的动力学描述符，如荧光变化率。
消耗NO和超氧阴离子的ONOO的生成导致对这些分子敏感的探针的细胞荧光强度降低。
这是一种快速且简单的检测方法，可用于在生理、病理和药理环境中监测细胞内ONOO的生成。© 2015国际临床细胞计量学会
