Template:SUIT text D074: Difference between revisions

Latest revision as of 16:48, 25 November 2021

SUIT-031 Q ce-pce D074 is a protocol to investigate simultaneously oxygen flux and the Q-redox state in the N- and NS-pathway and S-pathway control state in OXPHOS and in the S-pathway control state in the ET state in permeabilized cells. After permeabilization of the plasma membrane, coenzyme Q2 mimetic is titrated since the naturally occurring oQ 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.
The addition of PM leads to a partial reduction of CoQ₂ which is reflected in the increase of the Q-signal. ADP oxidizes CoQ₂, reflected in the decrease of the Q-signa. Addition of S initiates NS-OXPHOS capacity and results in further reduction of CoQ₂ in the OXPHOS state. Rotenone via CI inhibition reduces O₂ flux to S-linked OXPHOS capacity and causes oxidation of CoQ₂. 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 the full reduction of CoQ₂. This step is used as a reference step when calculating the Q redox 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-031 DLP files are currently provided for different applications. For O₂ application with ce-pce, choose [[]], for mt, choose SUIT-031. For Q redox state detection with mt, choose SUIT-031 Q mt D072.

How to measure the Q redox state, see: MiPNet24.12 NextGen-O2k: Q-Module

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-SUIT-031 Q ce-pce D074 is a protocol to investigate simultaneously oxygen flux and the redox state of the Q-pool in the [[NADH Electron transfer-pathway state| N-]] and [[NS-pathway control state| NS-pathway]] and [[S-pathway control state]] in OXPHOS and in the [[S-pathway control state]] in the ET state in permeabilized cells.  <br>
+SUIT-031 Q ce-pce D074 is a protocol to investigate simultaneously oxygen flux and the Q-redox state in the [[NADH Electron transfer-pathway state| N-]] and [[NS-pathway control state| NS-pathway]] and [[S-pathway control state]] in OXPHOS and in the [[S-pathway control state]] in the ET state in permeabilized cells. After permeabilization of the plasma membrane,  [[coenzyme Q2]] mimetic is titrated since the naturally occurring oQ is trapped in the mitochondrial inner membrane. 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 caused by the mimetics. Our study shows that 1 µM CoQ<sub>2</sub> was sufficient to detect the Q-redox change without an influence on respiration.  <br>
-After permeabilization of the plasma membrane,  [[coenzyme Q2]] mimetic was titrated since the naturally occurring CoQ is trapped in the mitochondrial inner membrane. CoQ2 reacts both with the biological site (CI, CII, and CIII) and the [[Q-Sensor]]. Application of the lowest possible CoQ2 concentration is recommended to avoid any side reaction on the ETS caused by the mimetics. Our study shows that 1 µM CoQ2 was sufficient to detect the redox change of the Q-pool without an influence on respiration.  <br>
+The addition of PM leads to a partial reduction of CoQ<sub>2</sub> which is reflected in the increase of the Q-signal. ADP oxidizes CoQ<sub>2</sub>, reflected in the decrease of the Q-signa. Addition of S initiates NS-OXPHOS capacity and results in further reduction of CoQ<sub>2</sub> in the OXPHOS state. Rotenone via CI inhibition reduces O<sub>2</sub> flux to S-linked OXPHOS capacity and causes oxidation of CoQ<sub>2</sub>. The [[uncoupler]] CCCP oxidizes CoQ<sub>2</sub>, 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>
-Addition of PM leads to partial reduction of the Q-pool which is reflected in the increase of the Q signal. ADP oxidizes the Q-junction, and the addition of S initiates NS-OXPHOS capacity and results in further reduction of the Q-pool in the OXPHOS state. This was followed by the addition of rotenone to detect S-linked OXPHOS capacity which is reflected in the oxidation the Q-pool and reduced the O2 flux
+Anoxia was reached when the mitochondria consumed 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 the full reduction of CoQ<sub>2</sub>. This step is used as a reference step when calculating the [https://wiki.oroboros.at/index.php/Q_redox_state Q redox 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.
-The use of rotenone, which inhibits Complex I, provides a specific coupling-control protocol for S-pathway, avoiding oxaloacetate inhibition towards succinate dehydrogenase and reverse electron transfer (RET) in the absence of ADP when the mt-membrane potential is high.  Furthermore, rotenone is needed to detect the fully oxidized Q state in the presence of sample and the [[coenzyme Q2]] mimetic but in the absence of ADP and succinate for calculating the [https://wiki.oroboros.at/index.php/Q_redox_state Q redox fraction].  <br>
+In the DatLab software, SUIT-031 DLP files are currently provided for different applications. For O<sub>2</sub> application with ce-pce, choose [[]], for mt, choose [[SUIT-031]]. For Q redox state detection with mt, choose [[SUIT-031 Q mt D072]].<br>
-Succinate leads to an almost full reduction of the Q-pool which is reflected in the increase of the Q signal. ADP slightly oxidizes the Q-junction, while oligomycin would further reduce the Q-pool. <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 Omy titration test. <br>
-The [[uncoupler]] CCCP oxidizes the Q-pool, however, using HEK293T cells (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 was reached when the cells consumed 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 the full reduction of the Q-pool. This step is used as a reference step when calculating the Q redox fraction. At the end of the protocol, the CIII inhibitor antimycin A can be added to check its effect on the fully reduced Q-pool 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 [https://wiki.oroboros.at/index.php/Export_DL-Protocol_User_(*.DLPU) DLPU] can be created. For O<sub>2</sub> application with ce-pce, choose [[SUIT-006 O2 ce-pce D029]] to create the DLPU, for mt, choose [[SUIT-006 O2 mt D047]]. For O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> measurements (AmR), choose [[SUIT-006 AmR mt D048]], and for O<sub>2</sub> 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 mt, choose [[SUIT-006 Q mt D071]].<br>
 How to measure the Q redox state, see: [[MiPNet24.12 NextGen-O2k: Q-Module]]