Description
Reference: A: protocol for simultaneous determination of O2 flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-034
SUIT number: D082_mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx;7Reox
O2k-Application: NADH
SUIT-034 NADH mt D082 allows the study of mitochondrial respiration and NADH fluorescence in two coupling control states, 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 NADH mt D084.
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-006 NADH mt D084, 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.
Communicated by Grings M, Cardoso Luiza HD (last update 2023-11-30)
Representative traces
File:SUIT-034 NADH mt D082.png File:SUIT-034 NADH mt D082.png
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
|
5Anox | N | CI | mt;1D.1;2PGM;3D2.5;4U;5Anox | |
6Myx | N | CI | mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx
| |
7Reox | ROX | mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx;7Reox |
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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
- SUIT-033 NADH mt D081: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Similar protocol without uncoupler titrations and ET state evaluation.
- SUIT-006 NADH mt D084: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Without titration of low concentration of ADP (0.1 ΞΌM) for depletion of endogenous substrates. Therefore, it is possible to measure LEAK respiration harmonizing it with SUIT-034_NADH_mt_D082.
- SUIT-034 O2 mt DXX: Control protocol for respiration only, allowing for cytochrome c test.
Compare SUIT protocols
Chemicals and syringes
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 | We do not recommend using any other uncoupler, e.g. Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U), due to the chemical background effect on fluorescence. |
5Anox | The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration. | |
6Myx | Myxothiazol | We do not recommend using any other inhibitor of complex III, e.g. Antimycin A (Ama), due to the chemical background effect on fluorescence. |
7Reox | Reoxygenation can be performed by opening the chamber, see: Open chamber. |
- Suggested stock concentrations are shown in the specific DL-Protocol.