Description
Reference: A: protocol for determination of O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-034
SUIT number: D082_mt;1D.1;2PGM;3D2.5;4U;5Myx
O2k-Application: O2
SUIT-034 O2 mt D111 is used as a control protocol for SUIT-034 NADH mt D082, allowing for cytochrome c test and the evaluation of mitochondrial respiration in OXPHOS and ET 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-006 02 mt D108.
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. The titration of a small concentration of ADP stimulates the depletion of the endogenous substrates, leading to a decrease in mitochondria respiration.
The addition of the complex III inhibitor myxothiazol allows the measurement of Rox.
Communicated by Grings M, Cardoso Luiza HD (last update 2024-01-12)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
mt | REN | mt
| ||
1D.1 | mt;1D.1
| |||
2PGM | PGM | N | CI | mt;1D.1;2PGM
|
3D2.5 | PGMP | N | CI | mt;1D.1;2PGM;3D2.5
|
4U | PGME | N | CI | mt;1D.1;2PGM;3D2.5;4U
|
5Myx | ROX | N | CI | mt;1D.1;2PGM;3D2.5;4U;5Myx
|
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- Coupling control
- Pathway control
- Β» Electron transfer pathway
- Β» Fatty acid oxidation pathway control state, F
- Β» NADH electron transfer-pathway state, N
- Β» Succinate pathway control state, S
- Β» NS-pathway control state, NS
- Β» Glycerophosphate pathway control state, Gp
- Β» Complex IV single step, CIV
- Β» Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- Β» Glutamate, G
- Β» Glycerophosphate, Gp
- Β» Malate, M
- Β» Octanoylcarnitine, Oct
- Β» Pyruvate, P
- Β» Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- Control protocol of SUIT-034 NADH mt D082 for respiration only, allowing for cytochrome c test.
- + Reasonable duration of the experiment.
- - When ATPases are present in the sample, it is not possible to reach the LEAK state.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors. The addition of liver homogenate is recommended in the washing protocol to bind strong inhibitors.
- After myxothiazol titration, this protocol can be extended with the Complex IV assay.
Compare SUIT protocols
- SUIT-034 NADH mt D082: Similar protocol with the possibility to perform simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria.
Chemicals and syringes
If the experiment is performed as a control for D082, we recommend using the same chemicals.
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1D.1 | ADP (D) | 40 mM stock solution. |
2PGM | Pyruvate (P), Glutamate (G), and Malate (M) | |
3D2.5 | ADP (D) | 500 mM stock solution. |
4U | SF6847 | |
6Myx | Myxothiazol |
- Suggested stock concentrations are shown in the specific DL-Protocol.