Template:SUIT text D081

SUIT-033 NADH mt D081 allows the study of mitochondrial respiration and NADH fluorescence in OXPHOS in the N-pathway. In the absence of ATPases in the sample, LEAK state can also be evaluated. In order to measure LEAK in the presence of ATPases, this protocol should be performed in parallel to SUIT-032 NADH mt D078.

After the addition of mitochondria in the absence of fuel substrates and ADP, Ren, respiration due to oxidation of endogenous substrates remaining after mitochondrial isolation is measured. For calibration of the fully oxidized NAD, this protocol contains a step with the titration of a small concentration of ADP, stimulating the depletion of the endogenous substrates and thus leading to accumulation of oxidized NAD. This protocol is used for cross-calibration of SUIT-032 NADH mt D078, where no low concentration of ADP is added before fuel substrates, allowing the measurement of LEAK respiration and NAD redox state.

Anoxia is reached by letting mitochondria fully consume the oxygen in the O2k-chambers. In the absence of O2, the ETS upstream of CIV is reduced and thus leads to an accumulation of reduced NAD. Under anoxia the complex III inhibitor myxothiazol is added and a further increase in the reduced NAD fraction can be observed. This step is then used for the calibration of the fully reduced NAD. At the end of the protocol, the reoxigenation of the chamber allows the measurement of Rox.