SUIT-001 O2 ce-pce D003: Difference between revisions
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|SUIT number=D003_ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd | |SUIT number=D003_ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd | ||
}} | }} | ||
::: '''[[MitoPedia: SUIT]]''' - '''[[SUIT reference protocol]] RP1 for | ::: '''[[MitoPedia: SUIT]]''' - '''[[SUIT reference protocol]] RP1 for cells that are permeabilized in the chamber''' | ||
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' FNSGp(Oct,PGM) | ::: '''[[Categories of SUIT protocols |SUIT-category]]:''' FNSGp(Oct,PGM) | ||
::: '''[[SUIT protocol pattern]]:''' orthogonal 1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp- | ::: '''[[SUIT protocol pattern]]:''' orthogonal 1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp- | ||
The SUIT-001 O2 ce-pce D003 protocol in combination with [[SUIT-002 O2 ce-pce D007]] is specially designed to provide a common reference for comparison of respiratory control of [[Permeabilized cells| permeabilized cells]] ( | The SUIT-001 O2 ce-pce D003 protocol in combination with [[SUIT-002 O2 ce-pce D007]] is specially designed to provide a common reference for comparison of respiratory control of [[Permeabilized cells| permeabilized cells]] (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of cell types. SUIT-001 O2 ce-pce D003 gives information of [[ROUTINE]] respiration (ce: non-permeabilized cells) and after the plasma membrane permeabilization (pce: permeabilized cells) of 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 pce D003 can be extended with the CIV assay module. | ||
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:::+ If NS- and FNS-pathways are similar (NS<sub>''E''</sub> ~ FNS<sub>''E''</sub>), the additivity index of N- and S-pathways in ET state can be evaluated. ย | :::+ If NS- and FNS-pathways are similar (NS<sub>''E''</sub> ~ FNS<sub>''E''</sub>), the additivity index of N- and S-pathways in ET state can be evaluated. ย | ||
:::+ This protocol can be extended with the Complex IV module. | :::+ This protocol can be extended with the Complex IV module. | ||
:::+ The addition of non-permeabilized cells to the chambers provides additional information ([[ROUTINE]] respiration) | :::+ The addition of non-permeabilized cells to the chambers provides additional information ([[ROUTINE]] respiration) which can not be obtained in [[SUIT-001 O2 mt D001]] where the cells added to the chambers are already permeabilized. | ||
:::- 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>. | ||
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::::* [[SUIT-002 O2 pce D007]] (RP2): Harmonized SUIT protocol for permeabilized cells. | ::::* [[SUIT-002 O2 pce D007]] (RP2): Harmonized SUIT protocol for permeabilized cells. | ||
::::* [[SUIT-001 O2 mt D001]]: SUIT-001 for permeabilized cells ( | ::::* [[SUIT-001 O2 mt D001]]: SUIT-001 for permeabilized cells (cells added to the chamber have been permeabilized before their addition). This SUIT protocol provides no information on [[ROUTINE]] respiration of intact cells (ce). | ||
::::* [[SUIT-004]]: Short version of RP1; without G, F and Gp. | ::::* [[SUIT-004]]: Short version of RP1; without G, F and Gp. |
Revision as of 14:44, 27 March 2019
Description
Abbreviation: FNSGp(Oct,PGM)
Reference: A Reference protocol RP1; ce: non-permeabilized cells, pce: permeabilized cells - SUIT-001
SUIT number: D003_ce1;1Dig;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
O2k-Application: O2
- MitoPedia: SUIT - SUIT reference protocol RP1 for cells that are permeabilized in the chamber
- SUIT-category: FNSGp(Oct,PGM)
- SUIT protocol pattern: orthogonal 1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp-
The SUIT-001 O2 ce-pce D003 protocol in combination with SUIT-002 O2 ce-pce D007 is specially designed to provide a common reference for comparison of respiratory control of permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol) in a wide variety of cell types. SUIT-001 O2 ce-pce D003 gives information of ROUTINE respiration (ce: non-permeabilized cells) and after the plasma membrane permeabilization (pce: permeabilized cells) of 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 pce D003 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) |
---|---|---|---|---|
ce1 | ROUTINE | ce1
| ||
1Dig | REN | ce1;1Dig
|
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 |
|
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
- 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-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 cells, SUIT-001 and SUIT-002 harmonize in: ROUTINE (ce1), 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 addition of non-permeabilized cells to the chambers provides additional information (ROUTINE respiration) which can not be obtained in SUIT-001 O2 mt D001 where the cells added to the chambers are already permeabilized.
- - The substrate combination for maximum ET capacity (FNSGP) is not obtained in SUIT-001 in favour of measuring SE.
- - Lengthy duration of the experiment.
Compare SUIT protocols
- SUIT-001 O2 pce D004: SUIT-001 O2 pce D003 (general SUIT-001 for permeabilized cells) differs from SUIT-001 O2 pce D004 (SUIT-001 for permeabilized PBMCs and PLTs) in the presence and absence of octanoylcarnitine, respectively.
- SUIT-002 O2 pce D007 (RP2): Harmonized SUIT protocol for permeabilized cells.
- SUIT-001 O2 mt D001: SUIT-001 for permeabilized cells (cells added to the chamber have been permeabilized before their addition). This SUIT protocol provides no information on ROUTINE respiration of intact cells (ce).
References
MitoPedia concepts: SUIT protocol, SUIT A, Find
MitoPedia methods:
Respirometry