Difference between revisions of "SUIT-001 O2 mt D001"
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{{MitoPedia | {{MitoPedia | ||
|abbr= | |abbr=RP1 mt | ||
|description=[[File:1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp | |description=[[File:Mt;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd .png|400px|SUIT-RP1]] | ||
|info= | |info=[[SUIT-001]] - '''[[SUIT reference protocol]] Reference protocol RP1 for isolated mitochondria, tissue homogenate and permeabilized cells''' | ||
|application=O2 | |application=O2 | ||
|SUIT number= | |SUIT number=D001_mt;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd | ||
}} | }} | ||
{{Template:SUIT text D001}} | |||
:::: [[ | __TOC__ | ||
:::: '''[[SUIT | Communicated by [[Doerrier C]], [[Gnaiger E]] (last update 2024-03-05) | ||
== Representative traces == | |||
[[File:D001_O2_traces.png|600px]] | |||
{{Template:SUIT-001}} | |||
== 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 [[Proficiency_test| MitoFit Proficiency test]] for inter-individual and inter-laboratory reproducibility quality control. | |||
:::* A succinate concentration of >10 mM may be required for saturating S<sub>''E''</sub> 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: SGp<sub>''E''</sub> (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 (NS<sub>''E''</sub> ~ FNS<sub>''E''</sub>), 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 S<sub>''E''</sub>. | |||
:::- Lengthy duration of the experiment. | |||
:::- We do not generally recommended the addition of permeabilized cells (already permeabilized), however 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 through [[Digitonin |digitonin]] addition. Therefore, SUIT-001 O2 ce-pce D003 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, tissue homogenate and permeabilized cells (already permeabilized). | |||
::::* [[SUIT-004]]: Short version of RP1; without G, F and Gp. | |||
::::* [[SUIT-006]]: Very short initial coupling control module. | |||
== Chemicals and syringes == | |||
{{Template:Chemicals SUIT-001}} | |||
{{Template:Chemicals CIV}} | |||
::: Suggested stock concentrations are shown in the specific DL-Protocol. | |||
== References == | == References == | ||
{{#ask:[[Category:Publications]] [[Additional label::SUIT- | {{#ask:[[Category:Publications]] [[Additional label::SUIT-001 O2 mt D001]] [[Additional label::SUIT-001]] | ||
| ?Was published in year=Year | | ?Was published in year=Year | ||
| ?Has title=Reference | | ?Has title=Reference | ||
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| order=descending | | order=descending | ||
}} | }} | ||
{{MitoPedia concepts | {{MitoPedia concepts | ||
|mitopedia concept=SUIT protocol | |mitopedia concept=SUIT protocol, SUIT A, Find | ||
}} | }} | ||
{{MitoPedia methods | {{MitoPedia methods | ||
|mitopedia method=Respirometry | |mitopedia method=Respirometry | ||
}} | }} | ||
Latest revision as of 18:31, 5 March 2024
Description
Abbreviation: RP1 mt
Reference: SUIT-001 - SUIT reference protocol Reference protocol RP1 for isolated mitochondria, tissue homogenate and permeabilized cells
SUIT number: D001_mt;1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
O2k-Application: O2
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 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 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, Gnaiger E (last update 2024-03-05)
Representative traces
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
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » 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.
- - We do not generally recommended the addition of permeabilized cells (already permeabilized), however 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 through digitonin addition. Therefore, SUIT-001 O2 ce-pce D003 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, tissue homogenate and permeabilized cells (already permeabilized).
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1PM | Pyruvate (P) and Malate (M) | |
2D | ADP (D) | |
2c | Cytochrome c (c) | |
3U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler |
4G | Glutamate (G) | |
5S | Succinate (S) | |
6Oct | Octanoylcarnitine (Oct) | |
7Rot | Rotenone (Rot) | |
8Gp | Glycerophosphate (Gp) | |
9Ama | Antimycin A (Ama) |
Step | Chemical(s) and link(s) | Comments |
---|---|---|
## AsTm | Ascorbate (As) and TMPD (Tm) | |
## Azd | Azide (Azd) |
- Suggested stock concentrations are shown in the specific DL-Protocol.
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, Mészáros 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