FIGURE 3.

Superoxide production in brain and RGC-5 mitochondria after complex I inhibition. Mitochondria isolated from brain (BR) and undifferentiated RGC-5 (uR5) cells were standardized for protein content, and 50 μL was 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 (GLU/MAL) and the complex I inhibitor rotenone (ROT). The mitochondrial superoxide production levels after the addition of glutamate/malate in brain 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.