From Bioblast
Description
Reference: A: protocol for simultaneous determination of O2 flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-006
SUIT number: D082_mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx;7Reox
O2k-Application: NADH
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
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.