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Difference between revisions of "SUIT-002"

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{{MitoPedia
{{MitoPedia
|abbr=FNSGp(Oct,PGM)
|abbr=RP2
|description=[[File:1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot-.png|400px]]
|description=[[File:1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot-.png|400px]]
|info='''A: RP2''' [[Doerrier 2018 Methods Mol Biol]] [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-002.pdf|Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:SUIT-002.pdf Versions]
|info='''A:''' [[Doerrier 2018 Methods Mol Biol]]
}}
}}
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' FNSGp(Oct,PGM)
::: '''[[SUIT protocol pattern]]:''' 1D;2M;3Oct;4M;5P;6G;7S;8Gp;9U;10Rot-
::: '''[[SUIT protocol pattern]]:''' diametral 1D;2M;3Oct;4M;5P;6G;7S;8Gp;9U;10Rot-


The SUIT-002 protocol in combination with [[SUIT-001]] provides a common reference for comparison of respiratory control of [[Mitochondrial preparations| mitochondrial preparations]] in a wide variety of species, tissues and cell types. SUIT-002 is specially designed to give information on [[Fatty_acid_oxidation_pathway_control_state|F-pathway]] in [[Oxidative phosphorylation|OXPHOS state]] avoiding FAO overestimation in the presence of [[Anaplerosis|anaplerotic]] pathways. Moreover, the pathway control in OXPHOS state ([[Fatty_acid_oxidation_pathway_control_state|F]], F(N), [[FN]], [[FNS]], [[FNSGp]] pathways) and in [[ET-capacity| ET state]] ([[FNSGp]] and [[SGp-pathway control state|SGp]]) can be evaluated by using this SUIT protocol. SUIT-002 can be extended with the CIV assay module.
The SUIT-002 protocol in combination with [[SUIT-001]] provides a common reference for comparison of respiratory control of [[Mitochondrial preparations| mitochondrial preparations]] in a wide variety of species, tissues and cell types. SUIT-002 is specially designed to give information on [[Fatty_acid_oxidation_pathway_control_state|F-pathway]] in [[Oxidative phosphorylation|OXPHOS state]] avoiding FAO overestimation in the presence of [[Anaplerosis|anaplerotic]] pathways. Moreover, the pathway control in OXPHOS state ([[Fatty_acid_oxidation_pathway_control_state|F]], F(N), [[FN]], [[FNS]], [[FNSGp]] pathways) and in [[ET capacity| ET state]] ([[FNSGp]] and [[SGp-pathway control state|SGp]]) can be evaluated by using this SUIT protocol. SUIT-002 can be extended with the CIV assay module.


__TOC__
__TOC__
  Communicated by [[Doerrier C]] and [[Gnaiger E]] (last update 2019-01-30)
  Communicated by [[Doerrier C]] and [[Gnaiger E]] (last update 2019-06-05)


== Specific SUIT protocols ==
== Specific SUIT protocols ==


=== SUIT-002 O2 mt D005 and SUIT-002 O2 pfi D006 ===
[[File:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|400px]]
[[File:1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|400px]]
[[File:D005_O2_traces.png|400px]]
[[File:D005_O2_traces.png|400px]]
*  [[SUIT-002 O2 mt D005]] for isolated mitochondria and tissue homogenate
*  [[SUIT-002 O2 mt D005]] for [[Isolated mitochondria|isolated mitochondria]], [[Tissue homogenate|tissue homogenate]] and [[Permeabilized cells|permeabilized cells]] (already permeabilized when they are added to the chamber) 
*  [[SUIT-002 O2 pfi D006]] for permeabilized fibers
*  [[SUIT-002 O2 pfi D006]] for [[Permeabilized_muscle_fibers|permeabilized muscle fibers]]
 
=== SUIT-002 O2 ce-pce D007 ===
[[File:ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|400px]]
[[File:ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|400px]]
[[File:D07_O2_traces.png|400px]]
[[File:D07_O2_traces.png|400px]]
*  [[SUIT-002 O2 pce D007]] for permeabilized cells
*  [[SUIT-002 O2 ce-pce D007]] for [[Living cells|living cells]] (ce) and [[Permeabilized cells|permeabilized cells]] (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)
[[SUIT-002 O2 pce D007a]] for permeabilized PBMCs and PLTs (absense of Oct)  


== References ==
=== SUIT-002 O2 ce-pce D007a ===
{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Protocol]] [[Additional label::SUIT-002]]
[[File:ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png|400px]]
|?Was published in year=Year
[[File:D07a_O2_traces.png|400px]]
|?Has title=Reference
[[SUIT-002 O2 ce-pce D007a]] for [[Living cells|living cells]] (ce) and [[Permeabilized cells|permeabilized PBMC and PLT]] (pce; permeabilization of the plasma membrane occurs into the chambers through digitonin addition)
|?Mammal and model=Organism
|?Tissue and cell=Tissue;cell
|format=broadtable
|limit=5000
|offset=0
|sort=Was published in year
|order=descending
}}


{{Template:SUIT-002}}
{{Template:SUIT-002}}
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:::+ SUIT-002 allows the depletion of endogenous substrates with ADP (1D).
:::+ SUIT-002 allows the depletion of endogenous substrates with ADP (1D).
:::+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.  
:::+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.  
:::+ F-pathway (3Oct-2M.1) compared to FN-pathway (5P) in OXPHOS state.
:::+ F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
:::+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp).
:::+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
:::+ Harmonization with [[SUIT-001]] allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statically used as repeat measurements.
:::+ Harmonization with [[SUIT-001]] allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
:::+ Harmonization with many SUIT protocols (up to step 7S).
:::+ Harmonization with many SUIT protocols (up to step 7S).
:::+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, ''P''/''E'' (8Gp/9U) at high ET capacity can be calculated.
:::+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, ''P''/''E'' (8Gp/9U) at high ET capacity can be calculated.
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== Compare SUIT protocols ==
== Compare SUIT protocols ==
::::* [[SUIT-002 O2 ce-pce D007]] (general SUIT-002 for permeabilized cells) differs from [[SUIT-002 O2 ce-pce D007]] (SUIT-002 for permeabilized PBMC and PLT) in the concentration and volume of the titrations.
::::* [[SUIT-002 O2 ce-pce D007]] for permeabilized cells (ROUTINE respiration in [[Living cells|living cells]] is obtained before selectively permeabilizing the plasma membrane).
::::* [[SUIT-002 O2 ce-pce D007a]] for permeabilized PBMC and PLT (ROUTINE respiration in [[Living cells|living cells]] is obtained before selectively permeabilizing the plasma membrane).


::::* [[SUIT-001]] (RP1): Harmonized SUIT protocol.
::::* [[SUIT-001]] (RP1): Harmonized SUIT protocol.
::::* [[SUIT-005]]
::::* [[SUIT-005]]
::::* [[SUIT-025]] SUIT-025 allows the evaluation of different ET-pathway states (F, F(N), FN, FNS and S) in OXPHOS state.
::::* [[SUIT-027]] SUIT-027 focuses on the analysis of the malate-anaplerotic pathway control state in mitochondrial preparations.
== References ==
{{#ask:[[Category:Publications]] [[Instrument and method::O2k-Protocol]] [[Additional label::SUIT-002]]
|?Was published in year=Year
|?Has title=Reference
|?Mammal and model=Organism
|?Tissue and cell=Tissue;cell
|format=broadtable
|limit=5000
|offset=0
|sort=Was published in year
|order=descending
}}


{{MitoPedia concepts
{{MitoPedia concepts

Revision as of 14:48, 8 June 2020


high-resolution terminology - matching measurements at high-resolution


SUIT-002

Description

1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot-.png

Abbreviation: RP2

Reference: A: Doerrier 2018 Methods Mol Biol

SUIT protocol pattern: 1D;2M;3Oct;4M;5P;6G;7S;8Gp;9U;10Rot-

The SUIT-002 protocol in combination with SUIT-001 provides a common reference for comparison of respiratory control of mitochondrial preparations in a wide variety of species, tissues and cell types. SUIT-002 is specially designed to give information on F-pathway in OXPHOS state avoiding FAO overestimation in the presence of anaplerotic pathways. Moreover, the pathway control in OXPHOS state (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be evaluated by using this SUIT protocol. SUIT-002 can be extended with the CIV assay module.

Communicated by Doerrier C and Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

SUIT-002 O2 mt D005 and SUIT-002 O2 pfi D006

1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png D005 O2 traces.png

SUIT-002 O2 ce-pce D007

Ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png D07 O2 traces.png

SUIT-002 O2 ce-pce D007a

Ce1;1Dig;1D;2M.1;3Oct;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama;12AsTm;13Azd.png D07a O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot-.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1D REN 1D
  • ADP is added to stimulate the consumption of endogenous fuel-substrates.


2M.1 1D;2M.1
3Oct OctMP F FAO 1D;2M.1;3Oct
  • Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
3c OctMcP F FAO 1D;2M.1;3Oct;3c
  • Respiratory stimulation of the FAO-pathway, F, by fatty acid, FA, in the presence of malate, M. Malate is a type N substrate (N), required for the F-pathway. The FA concentration has to be optimized to saturate the F-pathway, without inhibiting or uncoupling respiration.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
  • Addition of cytochrome c yields a test for integrity of the mtOM (cytochrome c control efficiency). Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (OXPHOS capacity P with saturating [ADP]).
4M2 OctMP F(N) FAO 1D;2M.1;3Oct;3c;4M2
5P OctPMP FN FAO&CI 1D;2M.1;3Oct;4M2;5P
  • Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
6G OctPGMP FN FAO&CI 1D;2M.1;3Oct;4M2;5P;6G
  • Respiratory stimulation by simultaneous action of the F-pathway and N-pathway with convergent electron flow in the FN-pathway for evaluation of an additive or inhibitory effect of F.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
7S OctPGMSP FNS FAO&CI&II 1D;2M.1;3Oct;4M2;5P;6G;7S
  • Respiratory stimulation by simultaneous action of the F-pathway, N-pathway, and S-pathway, with convergent electron flow in the FNS-pathway for reconstitution of TCA cycle function and additive or inhibitory effect of F.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
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
  • Respiratory stimulation by action of succinate and glycerophosphate, Gp, with convergent electron flow in the SGp-pathway (CII&GpDH-linked pathway to the Q-junction).
  • Noncoupled electron transfer state, ET state, with ET capacity E.
11Ama ROX 1D;2M.1;3Oct;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).


Questions.jpg


Click to expand or collaps

Strengths and limitations

  • SUIT-002 in combination with SUIT-001 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.
+ SUIT-002 allows the depletion of endogenous substrates with ADP (1D).
+ The protocol provides information on F-pathway in OXPHOS state. The low concentration of malate used in this protocol, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
+ F-pathway (3Oct-2M.1) can be compared to FN-pathway (5P) in OXPHOS state.
+ Pathway control in OXPHOS (F, F(N), FN, FNS, FNSGp pathways) and in ET state (FNSGp and SGp) can be observed.
+ Harmonization with SUIT-001 allows to perform both SUIT protocols in parallel. The cross-linked respiratory states can be statistically used as repeated measurements.
+ Harmonization with many SUIT protocols (up to step 7S).
+ In SUIT-002, the full set of pathways converging into Q (FNSGp) is obtained in OXPHOS and ET states. Therefore, P/E (8Gp/9U) at high ET capacity can be calculated.
+ This protocol can be extended with the Complex IV module.
- S-pathway in ET state is not obtained (it is obtained in SUIT-001).
- Lengthy duration of the experiment.


Compare SUIT protocols


  • SUIT-001 (RP1): Harmonized SUIT protocol.
  • SUIT-005
  • SUIT-025 SUIT-025 allows the evaluation of different ET-pathway states (F, F(N), FN, FNS and S) in OXPHOS state.
  • SUIT-027 SUIT-027 focuses on the analysis of the malate-anaplerotic pathway control state in mitochondrial preparations.

References

 YearReferenceOrganismTissue;cell
Doerrier 2018 Methods Mol Biol2018Doerrier 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_3Human
Mouse
Rat
Saccharomyces cerevisiae
Heart
Skeletal muscle
Endothelial;epithelial;mesothelial cell
Blood cells
HEK
Platelet


MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry