Use of spin traps to detect superoxide production in living cells by electron paramagnetic resonance (EPR) spectroscopy

•Spin trapping coupled to EPR allows specific detection of O2− in living cells.•A large variety of cyclic nitrone spin traps have been proposed for this purpose.•Different experimental procedures address production by detached or adherent cells.•Here we put together all recent advances, benefits, and limitations of the method.•We insist on necessary controls and importance of thorough data analysis. Detection of superoxide produced by living cells has been an on-going challenge in biology for over forty years. Various methods have been proposed to address this issue, among which spin trapping with cyclic nitrones coupled to EPR spectroscopy, the gold standard for detection of radicals. This technique is based on the nucleophilic addition of superoxide to a diamagnetic cyclic nitrone, referred to as the spin trap, and the formation of a spin adduct, i.e. a persistent radical with a characteristic EPR spectrum. The first application of spin trapping to living cells dates back 1979. Since then, considerable improvements of the method have been achieved both in the structures of the spin traps, the EPR methodology, and the design of the experiments including appropriate controls. Here, we will concentrate on technical aspects of the spin trapping/EPR technique, delineating recent breakthroughs, inherent limitations, and potential artifacts.