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Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence

de Campos, Richard P. S ; Siegel, Joseph M ; Fresta, Claudia G ; Caruso, Giuseppe ; da Silva, José A. F ; Lunte, Susan M

Analytical and bioanalytical chemistry, 2015-09, Vol.407 (23), p.7003-7012 [Periódico revisado por pares]

Berlin/Heidelberg: Springer Berlin Heidelberg

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Título:
Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence
Autor: de Campos, Richard P. S ; Siegel, Joseph M ; Fresta, Claudia G ; Caruso, Giuseppe ; da Silva, José A. F ; Lunte, Susan M
Assuntos: Analysis ; Analytical Chemistry ; Animals ; Biochemistry ; Biosensing Techniques - instrumentation ; Capillary Electrophoresis of Biomolecules ; Cell Survival - drug effects ; Cell Survival - physiology ; Characterization and Evaluation of Materials ; Chemical properties ; Chemistry ; Chemistry and Materials Science ; DNA ; Electrons ; Electrophoresis ; Electrophoresis, Microchip - instrumentation ; Equipment Design ; Equipment Failure Analysis ; fluorescence ; Fluorescent Dyes - chemical synthesis ; Food Science ; humans ; Hydrogen peroxide ; Inhibitors ; Joining ; Kinases ; Laboratory Medicine ; Laser induced fluorescence ; Lasers ; lipids ; Macrophages ; Metabolic Clearance Rate - drug effects ; Metabolic Clearance Rate - physiology ; Mice ; Microscopy, Fluorescence - instrumentation ; Monitoring/Environmental Analysis ; Nitric oxide ; Physiological aspects ; Physiology ; proteins ; Raw ; RAW 264.7 Cells ; reactive oxygen species ; Reactive Oxygen Species - metabolism ; Reproducibility of Results ; Research Paper ; Semiconductors ; Sensitivity and Specificity ; Sod ; Spectrometry, Fluorescence - instrumentation ; Stimulation ; Superoxide ; superoxide dismutase ; Superoxides - metabolism ; Systems Integration ; Tetradecanoylphorbol Acetate - analogs & derivatives ; Tetradecanoylphorbol Acetate - pharmacology
É parte de: Analytical and bioanalytical chemistry, 2015-09, Vol.407 (23), p.7003-7012
Notas: http://dx.doi.org/10.1007/s00216-015-8865-1
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Descrição: Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE⁺) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE⁺ per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE⁺ signal from possible interferences.
Editor: Berlin/Heidelberg: Springer Berlin Heidelberg
Idioma: Inglês

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Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence

de Campos, Richard P. S ; Siegel, Joseph M ; Fresta, Claudia G ; Caruso, Giuseppe ; da Silva, José A. F ; Lunte, Susan M

Berlin/Heidelberg: Springer Berlin Heidelberg

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