Difference between revisions of "Template:SUIT text D071"
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The SUIT-006 Q mt D071 is a coupling-control protocol for simultaneous measurement of O<sub>2</sub> flux and the Q-redox state | The SUIT-006 Q mt D071 is a coupling-control protocol for simultaneous measurement of O<sub>2</sub> 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. | 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 | Directly after the sample addition, [[coenzyme Q2]] mimetic is titrated. CoQ<sub>2</sub> 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 CoQ<sub>2</sub> concentration is recommended to avoid any side reactions on the ETS. Our study shows that 1 µM CoQ<sub>2</sub> was sufficient to detect the Q-redox change without an influence on respiration. <br> | ||
Rotenone, an inhibitor of Complex I, avoids oxaloacetate | Rotenone, an inhibitor of Complex I, avoids oxaloacetate accumulation which could inhibit [[succinate dehydrogenase]]. Furthermore, rotenone is needed to inhibit mitochondrial oxidation of residual endogenous substrates, allowing to detect the fully oxidized CoQ<sub>2</sub> in the presence of sample and the [[coenzyme Q2]] mimetic for calculating the [https://wiki.oroboros.at/index.php/Q_redox_state reduced Q fraction]. <br> | ||
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 | 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 reduction of CoQ<sub>2</sub> which is reflected in the increase of the Q-signal. <br> | ||
[[ADP]] | [[ADP]] titration in saturating concentration initiates the [[OXPHOS]] state, and increase in respiration is accompanied by slight oxidization of the Q-junction, reflected in a decrease of the Q-signal. <br> | ||
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 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 stepwise Omy titration. <br> | ||
The [[uncoupler]] CCCP | The titration of [[uncoupler]] such as CCCP leads to the [ET state]], and CoQ<sub>2</sub> may be further oxidized in parallel to increase in respiration, however, using mouse cardiac mitochondria (see figures) U did not influence either O<sub>2</sub> flux or the [[Q redox state]] which means that the respiration is not limited by the phosphorylation system in this sample type. <br> | ||
Anoxia | Anoxia is reached when the mitochondria fully consume the oxygen in the O2k-chambers. In the absence of O<sub>2</sub>, the ETS upstream of CIV is reduced and thus leads to full reduction of CoQ<sub>2</sub>. 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 CoQ<sub>2</sub> under anoxia. | ||
In the DatLab software, SUIT-006 DLP files are currently provided | In the DatLab software, SUIT-006 DLP files are currently provided with succinate as a substrate. For using this protocol with other substrate/inhibitor combinations, a personalized [https://wiki.oroboros.at/index.php/Export_DL-Protocol_User_(*.DLPU) DLPU] can be created. <br> | ||
On how to measure the Q-redox state, see: [[MiPNet24.12 NextGen-O2k: Q-Module]] | |||
Revision as of 15:09, 19 December 2023
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 is titrated. 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. 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 accumulation which could inhibit succinate dehydrogenase. Furthermore, rotenone is needed to inhibit mitochondrial oxidation of residual endogenous substrates, allowing to detect the fully oxidized CoQ2 in the presence of sample and the coenzyme Q2 mimetic 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 reduction of CoQ2 which is reflected in the increase of the Q-signal.
ADP titration in saturating concentration initiates the OXPHOS state, and increase in respiration is accompanied by slight oxidization of 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 stepwise Omy titration.
The titration of uncoupler such as CCCP leads to the [ET state]], and CoQ2 may be further oxidized in parallel to increase in respiration, 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 is reached when the 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 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 with succinate as a substrate. For using this protocol with other substrate/inhibitor combinations, a personalized DLPU can be created.
On how to measure the Q-redox state, see: MiPNet24.12 NextGen-O2k: Q-Module
