| Title |
Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence
|
|---|---|
| Published in |
Analytical & Bioanalytical Chemistry, July 2015
|
| DOI | 10.1007/s00216-015-8865-1 |
| Pubmed ID | |
| Authors |
Richard P. S. de Campos, Joseph M. Siegel, Claudia G. Fresta, Giuseppe Caruso, José A. F. da Silva, Susan M. Lunte |
| Abstract |
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. Graphical Abstract Indirect detection of intracellular superoxide production in macrophages using MitoHE and microchip electrophoresis with laser induced fluorescence detection. |
X Demographics
The data shown below were collected from the profile of 1 X user who shared this research output. Click here to find out more about how the information was compiled.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 1 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Scientists | 1 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 21 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Brazil | 1 | 5% |
| Unknown | 20 | 95% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Master | 5 | 24% |
| Student > Bachelor | 5 | 24% |
| Researcher | 3 | 14% |
| Student > Ph. D. Student | 2 | 10% |
| Other | 1 | 5% |
| Other | 1 | 5% |
| Unknown | 4 | 19% |
| Readers by discipline | Count | As % |
|---|---|---|
| Chemistry | 7 | 33% |
| Engineering | 3 | 14% |
| Medicine and Dentistry | 2 | 10% |
| Biochemistry, Genetics and Molecular Biology | 2 | 10% |
| Neuroscience | 2 | 10% |
| Other | 1 | 5% |
| Unknown | 4 | 19% |
Attention Score in Context
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#22,758,309
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Outputs from Analytical & Bioanalytical Chemistry
#7,541
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#236,669
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Outputs of similar age from Analytical & Bioanalytical Chemistry
#75
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