Detection of reactive oxygen species in primary cultures of cerebellar granule cells

The aim of this work was to develop a novel procedure useful to detect the formation of two reactive oxygen species, i.e. superoxide and singlet oxygen, in neuron monolayer primary cultures, thus, making possible the investigation of the effect of certain compounds on reactive oxygen species formation. Thus, use was made of two reactive oxygen species detecting systems consisting of ferricytochrome c (Fe-cyt c) and imidazole-RNO (N, N-dimethyl-4-nitrosoaniline) which allow for the photometric detection of superoxide anion and singlet oxygen, respectively. Both of them were used to assess the formation of reactive oxygen species in cerebellar granule cells exposed to glutamate: both superoxide anion and singlet oxygen proved to be generated in glutamate neurotoxicity in a way sensitive to glutamate NMDA-receptor inhibitor, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a, d)cyclohepten-5,10-imine hydrogen maleate), to Ca(2+) complexing agent, EGTA, and to certain antioxidants. In principle, the reported protocol can be applied to any cell type in culture.