Fig. 4.

Testing for the accumulation of H₂O₂ in the medium over time with muscle mitochondria. For A–C all assay components were added before beginning the assay, with the exception of Amplex Ultrared (AUR) which was added as indicated by vertical grey arrows (↑). Fluorescence is in arbitrary (rb.) units. Note, for A–C there is some initial fluorescent product due to the quality and age of the AUR that must be subtracted from all other values. (A) Negligible accumulation of H₂O₂ in medium with mitochondria respiring on 5 mM glutamate and malate. (B) Addition of 2 µM auranofin to mitochondria respiring on 5 mM glutamate and malate results in H₂O₂ accumulation. (C) Mitochondria respiring on 5 mM succinate accumulate H₂O₂ in the medium in a time dependent manner. Note, where the traces reach 1000 Arb. units the detector has reached its maximum for the sensitivity settings used. (D) Accumulation of H₂O₂ in the medium by mitochondria respiring on 5 mM succinate approaches a stable steady-state concentration. Data are mean±SEM (n=3).