Journal: Methods in cell biology

Article Title: Measurement of mitochondrial H 2 O 2 production under varying O 2 tensions

doi: 10.1016/bs.mcb.2019.12.008

Figure Lengend Snippet: Scheme of a routine experiment for the measurement of the dependence of H2O2 release on oxygen concentration, and data analysis. Representative traces are shown. The reaction is started with the addition of substrates, as shown by the arrow (S/G, 5 mM succinate and 1 mM glutamate) to the assay media containing 0.4 mg/mL mitochondria. The oxygen concentration is measured directly by the Oroboros respirometer (A); the fluorescence signal (B) corresponds to the amount of released H2O2 measured by optical module. The oxygen level decreases rapidly by continuously purging the headspace with argon (Ar). The increase in fluorescence intensity is recalculated to indicate the H2O2 concentration change (B, right) and further into the rate of H2O2 release (C), as detailed in the main text. Only 20 rate points at 20 fixed oxygen concentrations were used for fitting (D). The calculated rates of H2O2 release (circles) were plotted against oxygen concentration and fitted with a linear function Eq. (1) (D) to determine the slope (k) of the dependence. The slope of the dependence of H2O2 release rate on the oxygen concentration for different substrates (Mal+Pyr, 2 mM malate/5 mM pyruvate couple Suc + Glu, 5 mM succinate/2 mM glutamate couple; G3P, 40 mM glycerol 3-phosphate) in respiration state 2 or 3 or in the presence of 1 μM rotenone (E) is indicated. Note that the highest rate of ROS generation (k) was found during RET when succinate or glycerol 3-phosphate are oxidized.

Article Snippet: Instrumentation Mitochondrial respiration and H 2 O 2 release are measured using an Oroboros O2k high-resolution respirometer equipped with a two-channel fluorescence optical module (excitation at 525 nm and emission at 580–700 nm) to simultaneously monitor oxygen concentration and a change in fluorescence.

Techniques: Concentration Assay, Fluorescence

Journal: Methods in cell biology

Article Title: Measurement of mitochondrial H 2 O 2 production under varying O 2 tensions

doi: 10.1016/bs.mcb.2019.12.008

Figure Lengend Snippet:

Article Snippet: Instrumentation Mitochondrial respiration and H 2 O 2 release are measured using an Oroboros O2k high-resolution respirometer equipped with a two-channel fluorescence optical module (excitation at 525 nm and emission at 580–700 nm) to simultaneously monitor oxygen concentration and a change in fluorescence.

Techniques: Isolation, Fluorescence, Incubation