Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps

Abstract

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.

Keywords::

• EPR spectroscopy
• spin trapping
• RAW macrophages
• free radical
• CD-DIPPMPO

Acknowledgments

The authors wish to thank Dr. J.-C. Drapier (CNRS UPR 2301, Gif-sur-Yvette, France) for the generous gift of RAW 264.7 macrophages. They are also deeply grateful to Dr. A. Rockenbauer (Institute of Molecular Pharmacology, Research Centre for Natural Sciences, Budapest, Hungary) for the use of his simulation program and for his precious advice regarding the simulation of the ESR spectra; to Dr. J.-L. Boucher for stimulating discussions during the course of this work and for his critical reading of the manuscript; and to Drs. Y.-M. Frapart and D. Mansuy for their scientific guidance at the beginning of this work.

Declaration of interest

The authors are indebted to CNRS and the Agence Nationale de la Recherche (ANR-09-BLAN-0193-02, SPIN BioRad) for their financial support.

Supplementary material available online

Supplementary Figure 1 to be found online at http://informahealthcare.com/doi/abs/10.3109/10715762.2015.1045504.