SUIT-006 NADH mt D084: Difference between revisions
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ย Communicated byย [[Grings M]], [[Cardoso Luiza HD]] (last update 2023- | ย Communicated byย [[Grings M]], [[Cardoso Luiza HD]] (last update 2023-12-14) ย | ||
ย | |||
== Representative traces == | == Representative traces == | ||
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== Strengths and limitations == | == Strengths and limitations == | ||
ย | :::* SUIT-006 NADH mt D084ย in combination with [[SUIT-034 NADH mt D082]] provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows: | ||
ย | :::: 1. to obtain reference values. | ||
:::: 2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology. | |||
:::: 3. to screen specific defects. | |||
:::+ Reasonable duration of the experiment. | |||
:::- Fully oxidized [[NAD]] can only be obtained with the combination with [[SUIT-034 NADH mt D082]] or in the absence of endogenous substrates | |||
:::- 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 | |||
:::- The concentration of the oxidized and reduced NAD fraction cannot be determined. | |||
:::- Omy concentration has to be determined if used. Higher concentrations of Omy may inhibit the ET state. | |||
:::- Ama and CCCP cannot be used due to the high chemical background effect on fluorescence | |||
:::- CIV activity cannot be determined and cytochrome ''c'' test cannot be performed together with the [[NADH-Module]]. | |||
:::* Cytochrome ''c'' test can be performed in the following protocol:[[SUIT-006 O2 mt D022]]. | |||
:::* This protocol can be extended with the Complex IV module in the following protocol: [[SUIT-006 O2 mt D022]]. | |||
== Compare SUIT protocols == | == Compare SUIT protocols == |
Revision as of 17:54, 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 2023-12-14)
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
- SUIT-006 NADH mt D084 in combination with SUIT-034 NADH mt D082 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:
- 1. to obtain reference values.
- 2. to evaluate mitochondrial physiological diversity, generating a mt-database on comparative mitochondrial physiology.
- 3. to screen specific defects.
- + Reasonable duration of the experiment.
- - Fully oxidized NAD can only be obtained with the combination with SUIT-034 NADH mt D082 or in the absence of endogenous substrates
- - 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
- - The concentration of the oxidized and reduced NAD fraction cannot be determined.
- - Omy concentration has to be determined if used. Higher concentrations of Omy may inhibit the ET state.
- - Ama and CCCP cannot be used due to the high chemical background effect on fluorescence
- - CIV activity cannot be determined and cytochrome c test cannot be performed together with the NADH-Module.
- Cytochrome c test can be performed in the following protocol:SUIT-006 O2 mt D022.
- This protocol can be extended with the Complex IV module in the following protocol: SUIT-006 O2 mt D022.
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 and ET state evaluation.
- 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.