SUIT-032 NADH mt D078: Difference between revisions
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{{MitoPedia | {{MitoPedia | ||
|description=[[File:1mt;1PGM;2D;3Anox;4Myx;5Reox.png| | |description=[[File:1mt;1PGM;2D;3Anox;4Myx;5Reox.png|300px]] | ||
|info='''A''':ย protocol for simultaneous determination of O<sub>2</sub> flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- '''[[SUIT- | |info='''A''':ย protocol for simultaneous determination of O<sub>2</sub> flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- '''[[SUIT-032]]''' | ||
|application=NADH | |application=NADH | ||
|SUIT number=D078_File:1mt;1PGM;2D;3Anox;4Myx;5Reox | |SUIT number=D078_File:1mt;1PGM;2D;3Anox;4Myx;5Reox | ||
}} | }} | ||
{{ | {{Template:SUIT text D078}} | ||
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__TOC__ | __TOC__ | ||
ย Communicated byย [[Grings M]], [[Cardoso Luiza HD]] (last update 2023- | ย Communicated byย [[Grings M]], [[Cardoso Luiza HD]] (last update 2023-12-21) ย | ||
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:::+ H<sub><small>2</small></sub> gas from [[Oxia]] or N<sub><small>2</small></sub>/argon can be used to decrease O<sub><small>2</small></sub> concentration to obtain anoxia faster. | :::+ H<sub><small>2</small></sub> gas from [[Oxia]] or N<sub><small>2</small></sub>/argon can be used to decrease O<sub><small>2</small></sub> concentration to obtain anoxia faster. | ||
:::- Fully oxidized [[NAD]] can only be obtained with the combination with [[SUIT-033 NADH mt D081]] or with samples in which endogenous substrates are absent. | :::- Fully oxidized [[NAD]] can only be obtained with the combination with [[SUIT-033 NADH mt D081]] or with samples in which endogenous substrates are absent. | ||
:::- Careful washing is required after the experiment to avoid carry-over of | :::- Careful washing is required after the experiment to avoid carry-over of 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. ย | :::- The concentration of the oxidized and reduced NAD fraction cannot be determined. ย | ||
:::- Antimycin A and CCCP cannot be used due to the high chemical background effect on fluorescence. | :::- Antimycin A and CCCP cannot be used due to the high chemical background effect on fluorescence. | ||
:::- Cytochrome ''c'' test cannot be performed during the protocol as it affects fluorescence. Cytochrome ''c'' test can be performed in the following protocol: [[SUIT-032 O2 mt D109]]. | :::- Cytochrome ''c'' test cannot be performed during the protocol as it affects fluorescence. Cytochrome ''c'' test can be performed in the following protocol: [[SUIT-032 O2 mt D109]]. | ||
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== Compare SUIT protocols == | == Compare SUIT protocols == | ||
:::* [[SUIT- | :::* [[SUIT-006 NADH mt D084]]: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Similar protocol with uncoupler titrations and ET state evaluation. | ||
:::* [[SUIT-033 NADH mt D081]]: Protocol for simultaneous determination of O<sub><small>2</small></sub> flux and NADH autofluorescence in isolated mitochondria, allowing for calibration of the NAD redox ratios with samples that contain residual endogenous substrates. Additional titration of low concentration of ADP (0.1 ฮผM) for depletion of endogenous substrates and calibration of fully reduced NAD, allowing for cross-calibration with SUIT-032 NADH mt D078. | :::* [[SUIT-033 NADH mt D081]]: Protocol for simultaneous determination of O<sub><small>2</small></sub> flux and NADH autofluorescence in isolated mitochondria, allowing for calibration of the NAD redox ratios with samples that contain residual endogenous substrates. Additional titration of low concentration of ADP (0.1 ฮผM) for depletion of endogenous substrates and calibration of fully reduced NAD, allowing for cross-calibration with SUIT-032 NADH mt D078. | ||
:::* [[SUIT-032 O2 mt D109]]: Control protocol for respiration only, allowing for cytochrome ''c'' test. | :::* [[SUIT-032 O2 mt D109]]: Control protocol for respiration only, allowing for cytochrome ''c'' test. |
Latest revision as of 10:37, 12 January 2024
Description
Reference: A: protocol for simultaneous determination of O2 flux and NADH autofluorescence in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-032
SUIT number: D078_File:1mt;1PGM;2D;3Anox;4Myx;5Reox
O2k-Application: NADH
The coupling-control protocol SUIT-032 NADH mt D078 allows the study of mitochondrial respiration and NADH fluorescence in two coupling control states: LEAK, and OXPHOS in the N-pathway.
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. If these substrates are fully consumed by the mitochondria, this step can be used for an approximate calibration of oxidized NAD (NAD defined as the sum of the oxidized NAD+ and the reduced NADH). If this is not possible, this protocol should be used in combination with SUIT-033 NADH mt D081, where the titration of a small concentration of ADP leads to depletion of endogenous substrates, thus leading to accumulation of oxidized NAD, allowing to calibrate for the fully oxidized NAD.
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 2023-12-21)
Representative traces
File:SUIT-032 NADH mt D078 O2.png File:SUIT-032 NADH mt D078.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
|
3Anox | N | CI | 1PGM;2D;3Anox | |
4Myx | N | CI | 1PGM;2D;3Anox;4Myx
| |
5Reox | ROX | 1PGM;2D;3Anox;4Myx;5Reox |
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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-032 NADH mt D078 in combination with SUIT-033 NADH mt D081 provides NAD redox ratios in LEAK and OXPHOS states, measured simultaneously with respiration.
- + Reasonable duration of the experiment.
- + H2 gas from Oxia or N2/argon can be used to decrease O2 concentration to obtain anoxia faster.
- - Fully oxidized NAD can only be obtained with the combination with SUIT-033 NADH mt D081 or with samples in which endogenous substrates are absent.
- - Careful washing is required after the experiment to avoid carry-over of 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.
- - Antimycin A and CCCP cannot be used due to the high chemical background effect on fluorescence.
- - Cytochrome c test cannot be performed during the protocol as it affects fluorescence. Cytochrome c test can be performed in the following protocol: SUIT-032 O2 mt D109.
- After myxothyazol titration, this protocol can be extended with the Complex IV assay.
Compare SUIT protocols
- SUIT-006 NADH mt D084: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria. Similar protocol with uncoupler titrations and ET state evaluation.
- SUIT-033 NADH mt D081: Protocol for simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria, allowing for calibration of the NAD redox ratios with samples that contain residual endogenous substrates. Additional titration of low concentration of ADP (0.1 ฮผM) for depletion of endogenous substrates and calibration of fully reduced NAD, allowing for cross-calibration with SUIT-032 NADH mt D078.
- SUIT-032 O2 mt D109: Control protocol for respiration only, allowing for cytochrome c test.
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PGM | Pyruvate (P), Glutamate (G), and Malate (M) | |
2D | ADP (D) | |
3Anox | The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration. | |
4Myx | 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. |
5Reox | Reoxygenation can be performed by opening the chamber, see: Open chamber. |
- Suggested stock concentrations are shown in the specific DL-Protocol.