Template:SUIT text D071
The SUIT-006 Q mt D071 is a coupling-control protocol for simultaneous measurement of O2 flux and the Q-redox state in isolated mitochondria or permeabilized cells (which are permeabilized before adding them into the O2k-chamber). Different coupling control states are assessed (L(n) - P - E or L- P-L(Omy)- E) at the succinate-pathway control state. <be>
After the addition of mitochondria in the absence of fuel substrates and ADP, Rox with endogenous substrates (Ren) is detected due to oxidation of endogenous substrates remaining after mitochondrial isolation.
Directly after the sample addition, coenzyme Q2 mimetic was titrated since the naturally occurring CoQ is trapped in the mitochondrial inner membrane. CoQ2 reacts both with the mitochondrial complexes at the Q-binding site (CI, CII, and CIII) and with the detecting electrode of the Q-Sensor. Application of the lowest possible CoQ2 concentration is recommended to avoid any side reactions on the ETS caused by the mimetics. Our study shows that 1 µM CoQ2 was sufficient to detect the Q-redox change without an influence on respiration.
Rotenone, an inhibitor of Complex I, avoids oxaloacetate inhibition towards succinate dehydrogenase. Furthermore, rotenone is needed to detect the fully oxidized CoQ2 in the presence of sample and the coenzyme Q2 mimetic but in the absence of ADP and succinate for calculating the reduced Q fraction.
Succinate as a substrate of Complex II is oxidized to fumarate and supports electron transfer through CII to Q. Succinate with rotenone supports S-linked LEAK respiration and leads to an almost full reduction of CoQ2 which is reflected in the increase of the Q-signal.
ADP slightly oxidizes the Q-junction, reflected in a decrease of the Q-signal.
The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately. Since higher concentrations of Omy can decrease the ET state induced upon the addition of uncoupler, the required concentration of Omy has to be assessed by the Omy titration test.
The uncoupler CCCP oxidizes CoQ2, however, using mouse cardiac mitochondria (see figures) U did not influence either O2 flux or the Q redox state which means that the respiration is not limited by the phosphorylation system in this sample type.
Anoxia was reached when the mitochondria consumed the oxygen in the O2k-chambers. In the absence of O2, the ETS upstream of CIV is reduced and thus leads to full reduction of CoQ2. This step is used as a reference step when calculating the reduced Q fraction. At the end of the protocol, the CIII inhibitor antimycin A can be added to check its effect on the fully reduced CoQ2 under anoxia.
In the DatLab software, SUIT-006 DLP files are currently provided for the categories N(PM) and S. For using this protocol with other substrate/inhibitor combinations, a personalized DLPU can be created. For O2 application with ce-pce, choose SUIT-006 O2 ce-pce D029 to create the DLPU, for mt, choose SUIT-006 O2 mt D047. For O2 and H2O2 measurements (AmR), choose SUIT-006 AmR mt D048, and for O2 and membrane potential measurements, choose SUIT-006 Fluo mt D034, for ATP measurement (MgG) choose SUIT-006 MgG mt D055, and for Q redox state detection with ce-pce, choose SUIT-006 Q ce-pce D073.
How to measure the Q-redox state, see: MiPNet24.12 NextGen-O2k: Q-Module
