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Analytical and Bioanalytical Chemistry

, Volume 394, Issue 5, pp 1497–1504 | Cite as

Continuous electrochemical monitoring of nitric oxide production in murine macrophage cell line RAW 264.7

  • Michaela Pekarova
  • Jana Kralova
  • Lukas Kubala
  • Milan Ciz
  • Antonin Lojek
  • Cenek Gregor
  • Jan Hrbac
Original Paper

Abstract

In this study, we realized the continual and long-term electrochemical detection of NO production by stimulated macrophages using modified porphyrinic microsensor. The NO release from RAW 264.7 cells stimulated by lipopolysaccharide started 5 h after the lipopolysaccharide administration. After reaching its maximum at the sixth hour, the stable level of NO production was observed between the seventh and 12th hour of the experiment. This phase was followed by a gradual decline in NO production. A close correlation between the NO signal detected with microelectrode and nitrite accumulation, which had been determined in supernatants removed from stimulated cells, was observed. This finding was utilized for the calibration of the electrochemical experiment. The presence of iNOS enzyme, which constitutes a main requirement for NO production by stimulated macrophages, was confirmed by Western blot analysis of iNOS protein expression at key time points of the corresponding electrochemical experiment. The capability of our microsensor to instantaneously monitor the changes in the NO production by stimulated RAW 264.7 cells was demonstrated by the immediate decrease in the signal due to NO as a response to the addition of iNOS inhibitor into the cell culture medium.

Keywords

Nitric oxide Macrophages RAW 264.7 Nitric oxide sensor Nitrites iNOS expression 

Notes

Acknowledgment

This study was conducted under the research plans AVOZ50040507, AVOZ50040702, and 1M6198959201, supported from the Grant Agency of the Czech Republic (project no. 524/05/P135), the Grant Agency of the AS CR (no. 1QS 500040507), and MEYS (no. OC08058).

Supplementary material

216_2009_2813_MOESM1_ESM.pdf (205 kb)
ESM 1 (PDF 204 kb)

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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Michaela Pekarova
    • 1
  • Jana Kralova
    • 1
  • Lukas Kubala
    • 1
  • Milan Ciz
    • 1
  • Antonin Lojek
    • 1
  • Cenek Gregor
    • 2
  • Jan Hrbac
    • 2
  1. 1.Institute of BiophysicsAcademy of Sciences of the Czech RepublicBrnoCzech Republic
  2. 2.Department of Physical Chemistry, Faculty of SciencePalacky UniversityOlomoucCzech Republic

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