Superoxide Production in Cerebral, Neuroblastoma, and RGC-5 Mitochondria after Complex I Inhibition. Mitochondria isolated from cerebral, neuroblastoma, and undifferentiated RGC-5 cells were standardized for protein content and 50 μl were added to a well in a 96-well plate containing 50 μL Amplex Red (AR). Prior to the addition of solutions containing mitochondria a baseline of superoxide production was established by measuring the fluorescent product of H2O2 and AR, resorufin at approximately 10-second intervals. In a similar manner, production of superoxide was analyzed after the addition of the complex I substrates glutamate/malate and the complex I inhibitor rotenone. The mitochondrial superoxide production levels after the addition of glutamate/malate in cerebral cells were significantly higher than that of undifferentiated RGC-5 cells during basal metabolism, in the presence of glutamate/malate and after complex I inhibition with rotenone.