From Bioblast
Description
Reference: A: quality control evaluation of mitochondrial preparations - SUIT-029
MitoPedia concepts:
MiP concept,
SUIT protocol,
Recommended
MitoPedia methods:
Respirometry
- SUIT protocol pattern: 1PM;2T;3Omy;4U-
- The SUIT-029 protocol is specially designed to evaluate the quality of mitochondrial preparations in a wide variety of species, tissues and cell types. SUIT-029 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Using this protocol ATPase contamination and mitochondrial outer membrane integrity in different mitochondrial preparations with N substrates can be evaluated. In isolated mitochondria contaminated by ATPases, endogenous adenylates are phosphorylated to ATP in the LEAK state, which is recycled to ADP, which leads to an overestimation of LEAK respiration, when comparing L(Omy) and L(n). Therefore, it might be necessary to assess LEAK respiration in the presence of ATPases inhibitors, e.g. oligomycin. Optionally, additive effect of convergent electron flow through NADH- and NS-pathways can be evaluated in the ET state.
- The present SUIT-029 DLP file shows an application in with imt, thom and pce in the pathway category in N or NS. For using this protocol with other sample preparations or substrate/inhibitor combinations, a personalized DLPU can be created.
Communicated by Antunes D, Komlodi T, Gnaiger E (last update 2020-05-12)
Specific SUIT protocols
SUIT-029 O2 mt D066
- SUIT-029 O2 mt D066 for isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M)
|
---|---|---|---|---|
1PM | PML(n) | N | CI | 1PM
|
2T | PML or PMP | N | CI | 1PM;2T
|
2D | PMP | N | CI | 1PM;2T;2D
|
2c | PMcP | N | CI | 1PM;2T;2D;2c
|
3Omy | PMLOmy | N | CI | 1PM;2D;2c;3Omy
|
4U | PME | N | CI | 1PM;2T;2D;2c;3Omy;4U
|
5G | PGME | N | CI | 1PM;2T;2D;2c;3Omy;4U;5G
|
6S | PGMSE | NS | CI+CII | 1PM;2T;2D;2c;3Omy;4U;5G;6S
|
6U | PGMSE | NS | CI+CII | 1PM;2T;2D;2c;3Omy;4U;5G;6S;6U
|
7Rot | SE | S | CI | 1PM;2T;2D;2c;3Omy;4U;5G;6S;6U;7Rot
|
8Ama | ROX | 1PM;2T;2D;2c;3Omy;4U;5G;6S;6U;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
- This protocol allows to evaluate the quality of mitochondrial preparations taking into account the presence of ATPases.
- + PM is the superior alternative to GM: with GM the fraction of the N-pathway is lower and S-pathway contribution is higher compared to PM. PM, therefore, yields a more sensitive assay for the diagnosis of injuries in the N-pathway, since in the case of GM impairment N-pathway capacity can be compensated partially by activation of the S-pathway.
- + Evaluation of the ATPase contamination is included (2T).
- + Integrity of mitochondrial outer membrane is assessed ( 2c).
- + Overestimation of LEAK respiration is prevented in the presence of Oligomycin as a control with the L(n) previously obtained.
- + Evaluation of ET-state with NADH-linked substrates allows to calculate ET-coupling efficiency (1-L/E).
- + This protocol can be extended with the Complex IV module.
- - Long duration of the experiment due to adjustment of Oligomycin concentration (in the case of a new isolation protocol). For a shorter protocol the titration of glutamate, succinate and rotenone could be omitted.
- - The use of Oligomycin could affect the evaluation of the ET capacity (could inhibit ET-state).
- - Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
Compare SUIT protocols
- SUIT-001 (RP1): Harmonized SUIT protocol for isolated mitochondria, tissue homogenate and permeabilized cells.
- SUIT-006: Very short coupling protocol with PM as NADH-linked substrates.
- SUIT-008: A protocol to evaluate NS-linked pathway in the OXPHOS and ET states.
- SUIT-004: Short version of RP1; without G, F and Gp.
- SUIT-012: Coupling control with NADH-linked substrates (PM and PGM) without Omy addition.