MiPNet21.06 SUIT RP: Difference between revisions
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{{OROBOROS header page name}} | |||
{{Publication | {{Publication | ||
|title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. | |title=[[Image:O2k-Protocols.jpg|right|80px|link=O2k-Protocols|O2k-Protocols]] SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. | ||
|info=[ | |info=[''In progress''] | ||
|authors= | |authors=Oroboros | ||
|year= | |year=2018-06-25 | ||
|journal=Mitochondr Physiol Network | |journal=Mitochondr Physiol Network | ||
|abstract= | |abstract=Doerrier C, Sumbalova Z, Gnaiger (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(01):1-12. ย | ||
{{MiPNet pdf page linking to MitoPedia}} | |||
:ยป MitoPedia: [[SUIT reference protocol]] | :ยป MitoPedia: [[SUIT reference protocol]] | ||
:ยป Instrument: [[ | :ยป Instrument: [[Oroboros O2k]], [[Oroboros O2k-Catalogue | O2k-Catalogue]] | ||
|mipnetlab= | |mipnetlab=AT_Innsbruck_Oroboros | ||
}} | }} | ||
[[File:SUIT-catg FNSGpCIV.jpg|right|300px|SUIT-catg]] | [[File:SUIT-catg FNSGpCIV.jpg|right|300px|SUIT-catg]] | ||
== SUIT reference protocol RP1 == | |||
|- | ****:ยป [[SUIT-001|1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp-]] | ||
'''SUIT states:''' 1-3[[PM]](LPcE) 4[[PGM]] 5[[PGMS]] 6[[OctPGMS]] 7[[S]] 8[[SGp]] 9[[ROX]] 10[[Tm]] 11[[ROX]] | |||
{{Template:SUIT-001}} | |||
== SUIT reference protocol RP2 == | |||
****:ยป [[SUIT-002]] | |||
'''SUIT states:''' 1[[ROX]] 2[[OctM]](P) 2[[OctM]](c) 3[[OctPM]] 4[[OctPGM]] 5[[OctPGMS]] 6[[OctPGMSGp]] 7[[E]] 8[[SGp]] 9[[ROX]] 10[[CIV]] 11[[ROX]] | |||
{{Template:SUIT-002}} | |||
== Harmonized SUIT protocols == | |||
=== RP1 and RP2 in mt-preparations === | |||
| | '''SUIT RP1''' [[File:1PM;2D;2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd.png|400px|SUIT-RP1]] ย | ||
| | '''SUIT RP2''' [[File:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|500px|SUIT-RP2]] ย | ||
:::: '''SUIT RP1''':1PM;2D:2c;3U;4G;5S;6Oct;7Rot;'''8Gp''';9Ama;'''10AsTm''';11Azd | |||
:::: [[1D;2M;3Oct;4M;5P;6G;7S;8Gp;9U;10Rot-|SUIT RP2]]:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;'''10Rot''';11Ama;'''12AsTm''';13Azd | |||
| | |||
| | [[File:RP1&RP2.png|400px]] '''Harmonization between RP1 and RP2''' | ||
=== RP1 and RP2 in cells === | |||
RP1 and RP2 can be assessed in cells after the selective plasma membrane permeabilization. Digitonin is a mild detergent that permeabilizes plasma membranes. The optimum effective digitonin concentration for complete plasma membrane permeabilization of cultured cells can be determined directly in a respirometric protocol (see [[Digitonin]]). The use of living cells allows the possibility to evaluate respiration in two different modules: intact cells and permeabilized cells. ROUTINE respiration (ce1) is obtained before digitonin is employed, whereas after the use of digitonin we can address different [[Electron-transfer-pathway state]]s. | |||
:::: [[1PM;2D;3U;4G;5S;6Oct;7Rot;8Gp-#Harmonized_SUIT_protocols|SUIT RP1 in cells]]: '''ce1''';1Dig;1PM;2D:2c;3U;4G;5S;6Oct;7Rot;'''8Gp''';9Ama;'''10Tm''';11Azd | |||
| | :::: [[1D;2OctM;3P;4G;5S;6Gp;7U;8Rot-#Harmonized_SUIT_protocols|SUIT RP2 in cells]]: '''ce1''';1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;'''10Rot''';11Ama;'''12AsTm''';13Azd | ||
| | |||
| | [[File:Reference protocol RP1&RP2_pce.png|400px]] '''Harmonization between RP1 and RP2 in cells''' | ||
=== RP1 and RP2 in PBMC and PLT === | |||
In PBMC and PLT the reference protocol RP1 is performed in the absense of Oct: [[1PM;2D;3U;4G;5S;6Rot;7Gp-|SUIT 1 for PBMC and PTL]] | |||
| | |||
:::: '''SUIT RP1 for PBMC and PLT''': 1PM;2D;2c;3U;4G;5S;6Rot;'''7Gp''';8Ama;'''9AsTm''';10Azd | |||
:::: [[1D;2M;3Oct;4M;5P;6G;7S;8Gp;9U;10Rot-|SUIT RP2]]: 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;'''10Rot''';11Ama;'''12AsTm''';13Azd | |||
[[File:Reference protocol RP1&RP2_pce_PBMCs_PTLs.png|400px]] '''Harmonization between RP1 and RP2 in PBMC and PTL cells''' | |||
== Experimental details == | == Experimental details == | ||
Line 312: | Line 71: | ||
:::: Different concentrations of ADP are sufficient to obtain maximum flux for estimating OXPHOS capacity.ย The typical range for isolated mitochondria is 1 to 2.5 mM, for permeabilized cells 1 to 5 mM, for permeabilized muscle fibres 2.5 to 10 mM. | :::: Different concentrations of ADP are sufficient to obtain maximum flux for estimating OXPHOS capacity.ย The typical range for isolated mitochondria is 1 to 2.5 mM, for permeabilized cells 1 to 5 mM, for permeabilized muscle fibres 2.5 to 10 mM. | ||
=== RP-T01 === | === SUIT-RP development === | ||
ย | |||
==== RP-T01 ==== | |||
::::* RP1: Compared to RP1-T01, move Oct titration after S. | ::::* RP1: Compared to RP1-T01, move Oct titration after S. | ||
::::* RP2: Compared to RP2-T01, move U titration after Gp. | ::::* RP2: Compared to RP2-T01, move U titration after Gp. | ||
=== RP-T02 === | ==== RP-T02 ==== | ||
::::* RP2: Compared to RP2-T02, move D titration after the sample. Titration of M.1 before Oct. M2 is added after Oct. | ::::* RP2: Compared to RP2-T02, move D titration after the sample. Titration of M.1 before Oct. M2 is added after Oct. | ||
[[Talk:SUIT-2 O2 mt D05 |SUIT RP2 development]] | |||
=== Cytochrome c test === | === Cytochrome c test === | ||
Line 330: | Line 92: | ||
== Pre-publication communication == | == Pre-publication communication == | ||
:::: This communication is a pre-publication, inviting critical feedback and comments. All feedback will be carefully documented and evaluated in terms of justification of [[Gentle_Science#The_Vancouver_Protocol.2C_ICMJE.2C_and_Gentle_Science |co-authorship]]. We intend to finally publish this topic in a peer-reviewed journal as an original article, with reference to the pre-publication history including reviewer's reports. | :::: This communication is a pre-publication, inviting critical feedback and comments. All feedback will be carefully documented and evaluated in terms of justification of [[Gentle_Science#The_Vancouver_Protocol.2C_ICMJE.2C_and_Gentle_Science |co-authorship]]. We intend to finally publish this topic in a peer-reviewed journal as an original article, with reference to the pre-publication history including reviewer's reports. | ||
=== Experiments in progress === | === Experiments in progress === | ||
:::: The '''SUIT reference protocol''' is presently applied in permeabilized HEK cells, mouse heart isolated mitochondria, liver homogenate, permeabilized skeletal muscle (mouse and human), and human | :::: The '''SUIT reference protocol''' is presently applied in permeabilized HEK cells, mouse heart isolated mitochondria, liver homogenate, permeabilized skeletal muscle (mouse and human), and human PBMC and PLT. - [[User:Gnaiger Erich|Gnaiger Erich]] 18:47, 19 January 2016 (CET) | ||
[[Image:MitoFit.jpg|left|60px|link=http://www.mitofit.org/index.php/K-Regio MitoFit|K-Regio MitoFit]] | [[Image:MitoFit.jpg|left|60px|link=http://www.mitofit.org/index.php/K-Regio MitoFit|K-Regio MitoFit]] | ||
::::* [[AT Innsbruck Gnaiger E]] and [[AT Innsbruck | ::::* [[AT Innsbruck Gnaiger E]] and [[AT Innsbruck Oroboros]] - [[K-Regio MitoFit |MitoFit]] project. | ||
::::* [[US NC Winston-Salem Molina AJA]] - UPBEAT project. | ::::* [[US NC Winston-Salem Molina AJA]] - UPBEAT project. | ||
== Further details == | == Further details == | ||
::::ยป Introduction: [[Gnaiger 2014 MitoPathways]] | ::::ยป Introduction: [[Gnaiger 2014 MitoPathways]] | ||
::::ยป [[MitoPedia: SUIT]] | ::::ยป [[MitoPedia: SUIT]] | ||
::::* Table of titrations ยป [[MiPNet09.12 O2k-Titrations]] | ::::* Table of titrations ยป [[MiPNet09.12 O2k-Titrations]] | ||
::::ยป Definition: [[Substrate-uncoupler-inhibitor titration]] | ::::ยป Definition: [[Substrate-uncoupler-inhibitor titration]] | ||
::::ยป Context: [[SUIT protocol library]] | ::::ยป Context: [[SUIT protocol library]] | ||
::::ยป Abbreviations: [[MitoPedia]] | ::::ยป Abbreviations: [[MitoPedia]] | ||
{{Labeling | |||
|area=Instruments;methods | |||
|preparations=Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria | |||
|topics=Substrate | |||
|couplingstates=LEAK, OXPHOS, ET | |||
|pathways=F, N, S, Gp, CIV, NS, Other combinations | |||
|instruments=Oxygraph-2k | |||
|additional=MitoPathways, MitoFitPublication, SUIT-001, SUIT-002, SUIT-001 O2 mt D001, SUIT-001 O2 pfi D002, SUIT-001 O2 ce-pce D003, SUIT-001 O2 ce-pce D004, SUIT-002 O2 mt D005, SUIT-002 O2 pfi D006, SUIT-002 O2 ce-pce D007, SUIT-002 O2 ce-pce D007a}} |
Latest revision as of 10:52, 2 September 2022
MiPNet21.06 SUIT RP
SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. |
ยป [In progress]
Oroboros (2018-06-25) Mitochondr Physiol Network
Abstract: Doerrier C, Sumbalova Z, Gnaiger (2016) SUIT reference protocol for OXPHOS analysis by high-resolution respirometry. Mitochondr Physiol Network 21.06(01):1-12.
- ยป Current O2k-series: NextGen-O2k Series XB and O2k Series J
- ยป Current software versions DatLab 8.0: MitoPedia: DatLab
- Further details: ยป MitoPedia: O2k-Open Support
- ยป MitoPedia: SUIT reference protocol
- ยป Instrument: Oroboros O2k, O2k-Catalogue
โข O2k-Network Lab: AT_Innsbruck_Oroboros
SUIT reference protocol RP1
SUIT states: 1-3PM(LPcE) 4PGM 5PGMS 6OctPGMS 7S 8SGp 9ROX 10Tm 11ROX
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
- 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
SUIT reference protocol RP2
- ยป SUIT-002
SUIT states: 1ROX 2OctM(P) 2OctM(c) 3OctPM 4OctPGM 5OctPGMS 6OctPGMSGp 7E 8SGp 9ROX 10CIV 11ROX
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1D | REN | 1D
| ||
2M.1 | 1D;2M.1 | |||
3Oct | OctMP | F | FAO | 1D;2M.1;3Oct
|
3c | OctMcP | F | FAO | 1D;2M.1;3Oct;3c
|
4M2 | OctMP | F(N) | FAO | 1D;2M.1;3Oct;3c;4M2
|
5P | OctPMP | FN | FAO&CI | 1D;2M.1;3Oct;4M2;5P
|
6G | OctPGMP | FN | FAO&CI | 1D;2M.1;3Oct;4M2;5P;6G
|
7S | OctPGMSP | FNS | FAO&CI&II | 1D;2M.1;3Oct;4M2;5P;6G;7S
|
8Gp | OctPGMSGpP | FNSGp | FAO&CI&II&GpDH | 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp
|
9U | OctPGMSGpE | FNSGp | FAO&CI&II&GpDH | 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U
|
10Rot | SGpE | SGp | CII&GpDH | 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot
|
11Ama | ROX | 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama
|
- 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
Harmonized SUIT protocols
RP1 and RP2 in mt-preparations
- SUIT RP1:1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10AsTm;11Azd
- SUIT RP2:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd
Harmonization between RP1 and RP2
RP1 and RP2 in cells
RP1 and RP2 can be assessed in cells after the selective plasma membrane permeabilization. Digitonin is a mild detergent that permeabilizes plasma membranes. The optimum effective digitonin concentration for complete plasma membrane permeabilization of cultured cells can be determined directly in a respirometric protocol (see Digitonin). The use of living cells allows the possibility to evaluate respiration in two different modules: intact cells and permeabilized cells. ROUTINE respiration (ce1) is obtained before digitonin is employed, whereas after the use of digitonin we can address different Electron-transfer-pathway states.
- SUIT RP1 in cells: ce1;1Dig;1PM;2D:2c;3U;4G;5S;6Oct;7Rot;8Gp;9Ama;10Tm;11Azd
- SUIT RP2 in cells: ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd
Harmonization between RP1 and RP2 in cells
RP1 and RP2 in PBMC and PLT
In PBMC and PLT the reference protocol RP1 is performed in the absense of Oct: SUIT 1 for PBMC and PTL
- SUIT RP1 for PBMC and PLT: 1PM;2D;2c;3U;4G;5S;6Rot;7Gp;8Ama;9AsTm;10Azd
- SUIT RP2: 1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd
Harmonization between RP1 and RP2 in PBMC and PTL cells
Experimental details
Work in progress: pre-publication protocol
Saturating ADP concentrations
- Different concentrations of ADP are sufficient to obtain maximum flux for estimating OXPHOS capacity. The typical range for isolated mitochondria is 1 to 2.5 mM, for permeabilized cells 1 to 5 mM, for permeabilized muscle fibres 2.5 to 10 mM.
SUIT-RP development
RP-T01
- RP1: Compared to RP1-T01, move Oct titration after S.
- RP2: Compared to RP2-T01, move U titration after Gp.
RP-T02
- RP2: Compared to RP2-T02, move D titration after the sample. Titration of M.1 before Oct. M2 is added after Oct.
Cytochrome c test
- In protocol RP2, the cytochrome c test might better be moved from a c-titration after pyruvate (P) to a titration after M2. Otherwise, there is a risk of underestimation of FAO in cases of a cytochrome c effect. On the other hand, at the low flux before titration of P, the sensitivity of the cytochrome c test may be low.
- ~ Gnaiger Erich 18:26, 23 January 2016 (CET)
Mark names
- Even the highly abbreviated names of respiratory states have become too long for DatLab, where mark names are restricted to 8 digits. The visibility in DatLab of long mark names is restricted. The simplest solution for short mark names is the use of a numerical sequence, with the preceeding event name added for information.
- ~ Gnaiger Erich 16:50, 23 January 2016 (CET)
Pre-publication communication
- This communication is a pre-publication, inviting critical feedback and comments. All feedback will be carefully documented and evaluated in terms of justification of co-authorship. We intend to finally publish this topic in a peer-reviewed journal as an original article, with reference to the pre-publication history including reviewer's reports.
Experiments in progress
- The SUIT reference protocol is presently applied in permeabilized HEK cells, mouse heart isolated mitochondria, liver homogenate, permeabilized skeletal muscle (mouse and human), and human PBMC and PLT. - Gnaiger Erich 18:47, 19 January 2016 (CET)
- AT Innsbruck Gnaiger E and AT Innsbruck Oroboros - MitoFit project.
- US NC Winston-Salem Molina AJA - UPBEAT project.
Further details
- ยป Introduction: Gnaiger 2014 MitoPathways
- ยป MitoPedia: SUIT
- Table of titrations ยป MiPNet09.12 O2k-Titrations
- ยป Definition: Substrate-uncoupler-inhibitor titration
- ยป Context: SUIT protocol library
- ยป Abbreviations: MitoPedia
Labels: MiParea: Instruments;methods
Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria
Regulation: Substrate Coupling state: LEAK, OXPHOS, ET Pathway: F, N, S, Gp, CIV, NS, Other combinations HRR: Oxygraph-2k
MitoPathways, MitoFitPublication, SUIT-001, SUIT-002, SUIT-001 O2 mt D001, SUIT-001 O2 pfi D002, SUIT-001 O2 ce-pce D003, SUIT-001 O2 ce-pce D004, SUIT-002 O2 mt D005, SUIT-002 O2 pfi D006, SUIT-002 O2 ce-pce D007, SUIT-002 O2 ce-pce D007a