Description
Reference: A - SUIT-041
SUIT number: D096_1D;2M.1;3AC;3c;4M2;5P;6S;7Rot;8Ama
O2k-Application: O2
The SUIT-041 O2 mt D096 protocol provides a common reference for comparison of respiratory control of mitochondrial preparations such as isolated mitochondria, tissue homogenates and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species, tissues and cell types. SUIT-041 O2 mt D096 is specially designed to give information on F-pathway with titration of acylcarnitine as a FAO substrate to determine the optimal concentration. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS pathways) can be evaluated by using this SUIT protocol.
Communicated by Grings M, Cecatto C and Cardoso LHD (last update 2023-04-12)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1D | ROX | 1D
| ||
2M.1 | 1D;2M.1 | |||
3AC | ACMP | F | FAO | 1D;2M.1;3AC
|
3c | ACMcP | F | FAO | 1D;2M.1;3AC;3c
|
4M2 | ACMP | F(N) | FAO | 1D;2M.1;3AC;3c;4M2
|
5P | ACPMP | FN | FAO&CI | 1D;2M.1;3AC;4M2;5P
|
6S | ACPMSP | FNS | FAO&CI&II | 1D;2M.1;3AC;4M2;5P;6S
|
7Rot | SE | SGp | CII | 1D;2M.1;3AC;4M2;5P;6S;7Rot
|
8Ama | ROX | 1D;2M.1;3AC;4M2;5P;6S;7Rot;8Ama
|
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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
- SUIT-002 in combination with SUIT-001 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.
- SUIT-001 and SUIT-002 are used in the MitoFit Proficiency test for inter-individual and inter-laboratory reproducibility quality control.
- A succinate concentration of >10 mM may be required for saturating SE capacity.
- + SUIT-002 allows the depletion of endogenous substrates with ADP (1D).
- + The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
- + F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
- + Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
- + Harmonization with SUIT-001 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
- + Harmonization with many SUIT protocols (up to step 7S).
- + In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.
- + This protocol can be extended with the Complex IV module.
- - S-pathway in ET state is not obtained (it is obtained in SUIT-001).
- - Lengthy duration of the experiment.
- - We do not generally recommended the addition of already permeabilized cells, but we recommend to perform the permeabilization of the cell plasma membrane in the chambers (see SUIT-002 O2 ce-pce D007).
Compare SUIT protocols
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1D | ADP (D) | |
2M.1 | Malate (M) | |
3AC | Template:Acylcarnitine | |
3c | Cytochrome c (c) | |
4M2 | Malate (M) | |
5P | Pyruvate (P) | |
6S | Succinate (S) | |
7Rot | Rotenone (Rot) | |
8Ama | Antimycin A (Ama) |
- Suggested stock concentrations are shown in the specific DL-Protocol.