Analytical and Bioanalytical Chemistry

, Volume 407, Issue 23, pp 7003–7012 | Cite as

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

  • Richard P. S. de Campos
  • Joseph M. Siegel
  • Claudia G. Fresta
  • Giuseppe Caruso
  • José A. F. da Silva
  • Susan M. Lunte
Research Paper
Part of the following topical collections:
  1. Capillary Electrophoresis of Biomolecules


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


Bioanalytical methods Fluorescence Microchip electrophoresis Superoxide 

Supplementary material

216_2015_8865_MOESM1_ESM.pdf (524 kb)
ESM 1(PDF 524 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Richard P. S. de Campos
    • 1
    • 2
    • 3
    • 4
  • Joseph M. Siegel
    • 1
    • 2
  • Claudia G. Fresta
    • 1
    • 2
  • Giuseppe Caruso
    • 1
    • 2
  • José A. F. da Silva
    • 3
    • 4
  • Susan M. Lunte
    • 1
    • 2
    • 5
  1. 1.Ralph N. Adams Institute for Bioanalytical ChemistryUniversity of KansasLawrenceUSA
  2. 2.Department of ChemistryUniversity of KansasLawrenceUSA
  3. 3.Chemistry InstituteState University of CampinasCampinasBrazil
  4. 4.National Institute of Science and Technology in BioanalyticsINCTBioCampinasBrazil
  5. 5.Department of Pharmaceutical ChemistryUniversity of KansasLawrenceUSA

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