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: D109_mt;1PGM;2D;3Myx.png
O2k-Application: NADH
The coupling-control protocol SUIT-032 O2 mt D109 is used as a control protocol for SUIT-032 NADH mt D078, allowing for cytochrome c test and the evaluation of mitochondrial respiration 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.
The addition of the complex III inhibitor myxothiazol allows the measurement of Rox.
Communicated by Grings M, Cardoso Luiza HD (last update 2023-01-04)
Representative traces
File:SUIT-032 O2 mt D109 O2.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
|
3Myx | ROX | 1PGM;2D;3Myx
|
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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
- Control protocol of SUIT-032 NADH mt D078 for respiration only, allowing for cytochrome c test.
- + Reasonable duration of the experiment.
- - 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.
- - Omy concentration has to be determined if used. Higher concentrations of Omy may inhibit the ET state.
- 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 ΞΌM) for depletion of endogenous substrates and calibration of fully reduced NAD, allowing for cross-calibration with SUIT-032 NADH mt D078.
- SUIT-032 NADH mt D078: Similiar protocol with the possibility to perform simultaneous determination of O2 flux and NADH autofluorescence in isolated mitochondria.
Chemicals and syringes
If the experiment is performed as a control for D078, we recommend using the same chemicals.
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PGM | Pyruvate (P), Glutamate (G), and Malate (M) | |
2D | ADP (D) | |
3Myx | Myxothiazol |
- Suggested stock concentrations are shown in the specific DL-Protocol.