SUIT-001 O2 mt D001: Difference between revisions
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|SUIT number=D001_1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd | |SUIT number=D001_1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd | ||
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::: '''[[MitoPedia: SUIT]]''' - '''[[SUIT reference protocol]] RP1 for isolated mitochondria | ::: '''[[MitoPedia: SUIT]]''' - '''[[SUIT reference protocol]] RP1 for isolated mitochondria, tissue homogenate and permeabilized cells''' | ||
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' FNSGp(Oct,PGM) | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' FNSGp(Oct,PGM) | ||
::: '''[[SUIT protocol pattern]]:''' orthogonal 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp- | ::: '''[[SUIT protocol pattern]]:''' orthogonal 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp- | ||
The SUIT-001 O2 mt D001 protocol in combination with [[SUIT-002 O2 mt D005]] is specially designed to provide a common reference for comparison of respiratory control of isolated mitochondria | The SUIT-001 O2 mt D001 protocol in combination with [[SUIT-002 O2 mt D005]] is specially designed to provide a common reference for comparison of respiratory control of isolated mitochondria, tissue homogenates and permeabilized cells (the cells added to the chamber must be already permeabilized) in a wide variety of species and tissues. SUIT-001 O2 mt D001 gives information of the linear coupling control ([[LEAK-respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET-capacity| ''E'']]) with NADH linked-substrates ([[PM pathway control state|PM]]). Moreover, the pathway control in ET state ([[NADH_Electron_transfer-pathway_state|N]], [[NS-pathway control state| NS]], [[FNS]], [[Succinate pathway control state| S]] and [[SGp-pathway control state| SGp]] pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 mt D001 can be extended with the CIV assay module. | ||
__TOC__ | __TOC__ | ||
Β Communicated by [[Doerrier C]] and [[Gnaiger E]] (last update 2019-03- | Β Communicated by [[Doerrier C]] and [[Gnaiger E]] (last update 2019-03-27) | ||
{{Template:SUIT-001}} | {{Template:SUIT-001}} | ||
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:::- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring S<sub>''E''</sub>. | :::- The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring S<sub>''E''</sub>. | ||
:::- Lengthy duration of the experiment. | :::- Lengthy duration of the experiment. | ||
:::- The use of permeabilized cells (when the cells added to the chamber are already permeabilized) is not recommended. We recommend to perform the permeabilization of the cell plasma membrane into the chambers (see [[SUIT-001 O2 ce-pce D003]]).Β | |||
== Compare SUIT protocols == | == Compare SUIT protocols == | ||
Β Β | Β Β | ||
::::* [[SUIT-001 O2 ce-pce D003]]: SUIT-001 for non-permeabilized cells. Permeabilization of the cell plasma membrane occurs into the chambers after the addition of digitonin. Therefore, this SUIT protocol provides information of non-permeabilized cell respiration (ce) and permeabilized cell respiration (pce). | |||
::::* [[SUIT-002 O2 mt D005]] (RP2): Harmonized SUIT protocol for isolated mitochondria and tissue homogenate. | ::::* [[SUIT-002 O2 mt D005]] (RP2): Harmonized SUIT protocol for isolated mitochondria and tissue homogenate. | ||
Revision as of 12:12, 27 March 2019
Description
Abbreviation: FNSGp(Oct,PGM)
Reference: A Reference protocol RP1; mt: isolated mitochondria and tissue homogenate - SUIT-001
SUIT number: D001_1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
O2k-Application: O2
- MitoPedia: SUIT - SUIT reference protocol RP1 for isolated mitochondria, tissue homogenate and permeabilized cells
- SUIT-category: FNSGp(Oct,PGM)
- SUIT protocol pattern: orthogonal 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp-
The SUIT-001 O2 mt D001 protocol in combination with SUIT-002 O2 mt D005 is specially designed to provide a common reference for comparison of respiratory control of isolated mitochondria, tissue homogenates and permeabilized cells (the cells added to the chamber must be already permeabilized) in a wide variety of species and tissues. SUIT-001 O2 mt D001 gives information of the linear coupling control (L- P- E) with NADH linked-substrates (PM). Moreover, the pathway control in ET state (N, NS, FNS, S and SGp pathways) can be evaluated by using this SUIT protocol. SUIT-001 O2 mt D001 can be extended with the CIV assay module.
Communicated by Doerrier C and Gnaiger E (last update 2019-03-27)
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1PM | PML(n) | N | CI | 1PM
|
2D | PMP | N | CI | 1PM;2D
|
2c | PMcP | N | CI | 1PM;2D;2c
|
3U | PME | N | CI | 1PM;2D;3U
|
4G | PGME | N | CI | 1PM;2D;3U;4G
|
5S | PGMSE | NS | CI&II | 1PM;2D;3U;4G;5S
|
6Oct | OctPGMSE | FNS | FAO&CI&II | 1PM;2D;3U;4G;5S;6Oct
|
7Rot | SE | S | CII | 1PM;2D;3U;4G;5S;6Oct;7Rot
|
8Gp | SGpE | SGp | CII&GpDH | 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp
|
9Ama | ROX | 1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp;9Ama
|
Step | Respiratory state | Pathway control | ET-Complex | Comment |
---|---|---|---|---|
## AsTm | AsTmE | CIV | CIV | |
## Azd | CHB |
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- 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-001 in combination with SUIT-002 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.
- + Linear coupling control (L-P-E) with N substrates (PM).
- + Pathway control in ET state (N, NS, FNS, S and SGp pathways).
- + Harmonization with SUIT-002 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
- + In mtprep, SUIT-001 and SUIT-002 harmonize in: SGpE (8Gp in SUIT-001 and 10Rot in SUIT-002) and Complex IV.
- + Harmonization with many SUIT protocols (up to step 5S).
- + Oct is added in ET state to avoid uncoupler effect with fatty acids.
- + If NS- and FNS-pathways are similar (NSE ~ FNSE), the additivity index of N- and S-pathways in ET state can be evaluated.
- + This protocol can be extended with the Complex IV module.
- - The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring SE.
- - Lengthy duration of the experiment.
- - The use of permeabilized cells (when the cells added to the chamber are already permeabilized) is not recommended. We recommend to perform the permeabilization of the cell plasma membrane into the chambers (see SUIT-001 O2 ce-pce D003).
Compare SUIT protocols
- SUIT-001 O2 ce-pce D003: SUIT-001 for non-permeabilized cells. Permeabilization of the cell plasma membrane occurs into the chambers after the addition of digitonin. Therefore, this SUIT protocol provides information of non-permeabilized cell respiration (ce) and permeabilized cell respiration (pce).
- SUIT-002 O2 mt D005 (RP2): Harmonized SUIT protocol for isolated mitochondria and tissue homogenate.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet21.06 SUIT RP | 2018-06-25 | SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. | ||
Doerrier 2018 Methods Mol Biol | 2018 | Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, MΓ©szΓ‘ros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3 | Human Mouse Rat Saccharomyces cerevisiae | Heart Skeletal muscle Endothelial;epithelial;mesothelial cell Blood cells HEK Platelet |
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry