SUIT-006 NADH mt D084: Difference between revisions
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
:::* [[SUIT-032 NADH mt D078]]: Coupling-control protocol for simultaneous determination of O2 flux and NADH autofluorescence in mt-preparations (mtprep). Similar protocol without uncoupler titrations. | |||
:::* [[SUIT-034 NADH mt D082]]: Coupling-control protocol for simultaneous determination of O2 flux and NADH autofluorescence in mt-preparations (mtprep). Additional titration of low concentration of ADP (0.1 ฮผM) for depletion of endogenous substrates and calibration of fully reduced NAD. Cross-calibration with SUIT-006 NADH mt D084 | |||
== Chemicals and syringes == | == Chemicals and syringes == |
Revision as of 17:43, 14 December 2023
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: D084_mt;1PGM;2D;3(Omy);4U;5Anox;6Myx;7Reox
O2k-Application: NADH
Communicated by Grings M, Cardoso Luiza HD (last update 2023-11-30)
Representative traces
File:SUIT-006 NADH mt D084 O2.png File:SUIT-006 NADH mt D084.png
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
mt | REN | mt | ||
1PGM | PGML(n) | N | CI | 1PGM
|
2D | PGMP | N | CI | 1PGM;2D
|
(3Omy) | PGML(Omy) | N | CI | 1PGM;2D;(3Omy)
|
4U | PGME | N | CI | 1PGM;2D;(3Omy);4U
|
5Anox | N | CI | 1PGM;2D;(3Omy);4U;5Anox | |
6Myx | N | CI | 1PGM;2D;(3Omy);4U;5Anox;6Myx
| |
7Reox | ROX | 1PGM;2D;(3Omy);4U;5Anox;6Myx;7Reox |
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- Coupling control
- Pathway control
- ยป Electron transfer pathway
- ยป Fatty acid oxidation pathway control state, F
- ยป NADH electron transfer-pathway state, N
- ยป Succinate pathway control state, S
- ยป NS-pathway control state, NS
- ยป Glycerophosphate pathway control state, Gp
- ยป Complex IV single step, CIV
- ยป Anaplerotic pathway control state
- Pathway control
- Main fuel substrates
- ยป Glutamate, G
- ยป Glycerophosphate, Gp
- ยป Malate, M
- ยป Octanoylcarnitine, Oct
- ยป Pyruvate, P
- ยป Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
Compare SUIT protocols
- SUIT-032 NADH mt D078: Coupling-control protocol for simultaneous determination of O2 flux and NADH autofluorescence in mt-preparations (mtprep). Similar protocol without uncoupler titrations.
- SUIT-034 NADH mt D082: Coupling-control protocol for simultaneous determination of O2 flux and NADH autofluorescence in mt-preparations (mtprep). Additional titration of low concentration of ADP (0.1 ฮผM) for depletion of endogenous substrates and calibration of fully reduced NAD. Cross-calibration with SUIT-006 NADH mt D084
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PGM | Pyruvate (P), Glutamate (G), and Malate (M) | |
2D | ADP (D) | |
(3Omy) | Oligomycin (Omy) | This step can be skipped. |
4U | SF6847 | We do not recommend the use of any other uncoupler, like 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 the use of any other inhibitor of complex III, like 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.