Concomitant real-time monitoring of intracellular reactive oxygen species and mitochondrial membrane potential in individual living promonocytic cells

• Published in: Journal of immunological methods 2006-10, Vol.316 (1), p.27-41
• Main Authors: Zurgil, Naomi, Shafran, Yana, Afrimzon, Elena, Fixler, Dror, Shainberg, Asher, Deutsch, Mordechai
• Format: Article
• Language: English
• Subjects: 2,7-dichlorofluorescein diacetate; Biological and medical sciences; carbonyl cyanide m-chloro-phenyl hydrazone; CCCP; DCF-DA; DHE; DHR123; Dicarbethoxydihydrocollidine - analogs & derivatives; Dicarbethoxydihydrocollidine - blood; dihydroethidium; dihydrorhodamine123; FLT; Fluoresceins - chemistry; fluorescence intensity; fluorescence lifetime; fluorescence polarization; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Hydrogen Peroxide - pharmacology; IBC; ICS; Individual blood cells; individual cell scanner; L-NAME; LDL; lipopolysaccharide; Lipopolysaccharides - pharmacology; Live Cell Array (LCA); low-density lipoproteins; LPC; LPS; lysophosphatidylcholine; Membrane Potentials; Microscopy, Fluorescence; Microscopy, Polarization; mitochondrial membrane potential; Mitochondrial membrane potential (MMP); Mitochondrial Membranes - metabolism; Mitochondrial Membranes - physiology; MMP; Molecular immunology; Nω-nitro- larginine methyl ester; OLCA; Optical LiveCell; propidium iodide; reactive nitrogen species; Reactive oxygen species; Reactive oxygen species (ROS); Reactive Oxygen Species - analysis; Reactive Oxygen Species - metabolism; Rhodamine 123 - chemistry; RNS; ROS; SOD; superoxide dismutase; Techniques; tetramethylrhodamine methyl ester; Tissue Array Analysis - methods; TMRM; U937 Cells
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2006.07.013

Reactive oxygen species (ROS) have recently been shown to be involved in multiple physiological responses through modulation of signaling pathways. Inappropriate production of these radicals, and their metabolites, leads to the development of various pathologies. Free radicals can induce both positive and negative effects in cells, and their metabolic pathways are very complex. Hence, it is crucial to be able to simultaneously and directly determine their production dynamics and concentrations in individual living cells, in physiological or pathological states, and in response to drugs. The aim of the present study was to monitor in real time the rates of ROS generation in promonocytic cells upon stimulation with hydrogen peroxide and oxidized lipid. Quantitative detection of intracellular ROS concentration in intact living U937 cells was performed by fluorescence intensity (FI) and polarization (FP) measurements utilizing the Optical LiveCell™ Array technology. The “dihydro” derivative probes of fluorescein (DCF-DA) and rhodamine (DHR123) were used to assess the intracellular levels of ROS. Each probe molecule exhibited a characteristic FI and FP in its non-fluorescent or oxidized form. Analysis of the temporal relationship between the kinetics of ROS generation and the onset of changes in mitochondrial membrane potential shows high variability within a cell population with regard to both processes. The data demonstrated that temporal measurement of ROS generation, in identifiable individual cells, reveals kinetic behavior that otherwise would be undetected.