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Difference between revisions of "SUIT-006"

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{{MitoPedia
{{MitoPedia
|abbr=N, S, F, Gp
|abbr=[[Coupling control protocol |CCP]]
|description=[[File:1X;2D;2c;3Omy;4U;5Ama.png|450px]]
|description=[[File:1X;2D;2c;3Omy;4U;5Ama.png|450px]]
|info='''A: Coupling control protocol, [[Mitochondrial preparations |mtprep]]''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-006.pdf|Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:SUIT-006.pdf Versions]
|info='''A: [[Coupling control protocol]], [[Mitochondrial preparations |mtprep]]'''
}}
}}
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' N, S, F, Gp
::: '''[[SUIT protocol pattern]]:''' 1X;2D;3Omy;4U;5Ama
::: '''[[SUIT protocol pattern]]:''' linear [[coupling control protocol]] 1X;2D;3Omy;4U;5Ama
The SUIT-006 protocol is a [[coupling control protocol]] for [[mitochondrial preparations]]. Depending on the aims of the researcher, different combinations of substrates can be used to assess coupling control ([[LEAK-respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET-capacity| ''E'']] or [[LEAK-respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[LEAK-respiration|''L''(Omy)]]-[[ET-capacity| ''E'']]) at a defined [[electron transfer-pathway state]]. The use of [[oligomycin]] is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated [[LEAK]] respiration measured in the absence of adenylates - [[LEAK state without adenylates|L(n)]]. Therefore, [[oligomycin]] can be used to verify whether this occurs and obtain the [[LEAK]] state appropriately.
The SUIT-006 protocol is a [[coupling control protocol]] for [[mitochondrial preparations]]. Depending on the aims of the researcher, different combinations of substrates can be used to assess coupling control ([[LEAK-respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[ET-capacity| ''E'']] or [[LEAK-respiration|''L'']]-[[Oxidative phosphorylation| ''P'']]-[[LEAK-respiration|''L''(Omy)]]-[[ET-capacity| ''E'']]) at a defined [[electron transfer-pathway state]]. The use of [[oligomycin]] is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated [[LEAK]] respiration measured in the absence of adenylates - [[LEAK state without adenylates|L(n)]]. Therefore, [[oligomycin]] can be used to verify whether this occurs and obtain the [[LEAK]] state appropriately.


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  Communicated by [[Cardoso LH]], [[Doerrier C]], [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-04-02)
  Communicated by [[Cardoso LH]], [[Doerrier C]], [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-06-05)


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

Revision as of 11:37, 5 June 2019


high-resolution terminology - matching measurements at high-resolution


SUIT-006

Description

1X;2D;2c;3Omy;4U;5Ama.png

Abbreviation: CCP

Reference: A: Coupling control protocol, mtprep

SUIT protocol pattern: 1X;2D;3Omy;4U;5Ama

The SUIT-006 protocol is a coupling control protocol for mitochondrial preparations. Depending on the aims of the researcher, different combinations of substrates can be used to assess coupling control (L- P- E or L- P-L(Omy)- E) at a defined electron transfer-pathway state. The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately.

In the DatLab software, SUIT-006 DLP files are currently provided for the categories N(PM) and S. For using this protocol with other substrate/inhibitor combinations, a personalized DLPU can be created. For O2 application with ce-pce, choose SUIT-006_O2_ce-pce_D029 to create the DLPU, for mt, choose SUIT-006 O2 mt D047. For O2 and H2O2 measurements (AmR), choose SUIT-006 AmR mt D048, and for O2 and membrane potential measurements, choose SUIT-006 Fluo mt D034.


Communicated by Cardoso LH, Doerrier C, Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

SUIT-006 O2 ce-pce D029

Ce1;1Dig;1PM;2D;2c;3Omy;4U;5Ama.png SUIT-006 O2 pce D029 traces.png

  • SUIT-006 O2 ce-pce D029 for N-pathway with permeabilized cells. Omy may be added in order to not overestimate LEAK respiration, that might be overestimated in 1PM if there are endogenous adenylates.

SUIT-006 O2 mt D047

1PM;2D;2c;3Omy;4U;5Ama.png SUIT-006 O2 mt D047.png

  • SUIT-006 O2 mt D047 for N-pathway with isolated mitochondria or tissue homogenate. Omy may be added in order to not overestimate LEAK respiration, that might be overestimated in 1PM if there are endogenous adenylates.

SUIT-006 AmR mt D048

1PM;2D;3Omy;4U;5Ama.png SUIT-006 AmR mt D048 O2.png SUIT-006 AmR mt D048.png

  • SUIT-006 AmR mt D048 to study simultaneously the O2 flux and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells, in particular to study the influence of mt-membrane potential on NADH-linked pathway initiated mt-H2O2 production.

SUIT-006 Fluo mt D034

1PM;2D;3Omy;4U;5Ama.png SUIT-006 Fluo mt D034 O2.png SUIT-006 Fluo mt D034.png

  • SUIT-006 Fluo mt D034 to study simultaneously the O2 flux and mitochondrial membrane potential on isolated mitochondria, tissue homogenate and permeabilized cells.

SUIT-006 MgG mt D055

1PM;2D;3Cat;4U;5Ama.png SUIT-006 MgG mt D055 O2.png SUIT-006 MgG mt D055.png

  • SUIT-006 MgG mt D055 to study simultaneously the O2 flux and mitochondrial ATP production on isolated mitochondria, tissue homogenate and cells permeabilized previouslyto addition to the O2k chamber.

SUIT-006 O2 mt D022

1SRot;2D;2c;3(Omy);4U;5Ama.png SUIT-006 O2 mt D022 traces.png

  • SUIT-006 O2 mt D022 for S-pathway with isolated mitochondria, tissue homogenate and permeabilized cells.


MitoPedia: SUIT

Steps and respiratory states

1X;2D;2c;3Omy;4U;5Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1X XL(n) 1X
  • X: combination of substrates and inhibitors chosen according to the aims.
  • Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D XP 1X;2D
2c XcP 1X;2D;2c
  • 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]).
3Omy XLOmy 1X;2D;2c;3Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin is optional depending on the experimental question to resolve. If it is known that Ln has the same values for LOmy for a kind of sample and substrates, this step may be skipped. If the carrier of oligomycin (EtOH) is used instead, it is possible to evaluate whether it affects OXPHOS. In these cases, LEAK state is not reached again.
4U XE 1X;2D;2c;3Omy;4U
5Ama ROX 1X;2D;2c;3Omy;4U;5Ama
  • 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

+ Different combinations of substrates and inhibitors (X) can be used depending on the specific aims, e.g.: PGM, PM, GM, MnaPM or other combinations for N-pathway control state; S or RotS for S-pathway control state.
+ The combinations of substrates for one ET-pathway state with inhibitors for other pathways provide the best coupling control protocols for one specific ET-pathway state, e.g.: PM in the presence of Mna for N-pathway control state, succinate in the presence of rotenone for S-pathway control state. In the specific case of S-pathway control state, rotenone should be added to avoid reverse electron transfer (RET).
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module.
- This protocol does not include internal ET-pathway control steps.
- Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors, if applied (e.g., rotenone&succinate, SRot-pathway control state).
- LEAK respiration in the absence of Omy may be overestimated due to ATP turnover stimulated by endogenous adenylates. This can be evaluated by SUIT 1X;2D;3Omy;4U;-. In mouse brain homogenate with succinate&rotenone, L(n) and L(Omy) are identical (Krumschnabel et al 2014).
- CIV activity and Cytochrome c test cannot be performed together with the fluorescence (SUIT-006 AmR mt D048, SUIT-006 AmR mt D034 and SUIT-006 MgG mt D055).

Compare SUIT protocols

  • SUIT-001: Extended protocol (Reference protocol 1) with SUIT-006 as the initial coupling control module (with PM as NADH-linked substrates).
  • SUIT-004: Shortened version of SUIT-001, also with SUIT-006 as the initial coupling control module (with PM as NADH-linked substrates).
  • SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).

References

 YearReferenceOrganismTissue;cell
MiPNet26.10 MgG data analysis2021-09-16
O2k-Protocols
Measurement of mitochondrial ATP production using Magnesium Green: DL-Protocols and data analysis with DatLab 7.4
Komlodi 2021 BEC AmR-O22021Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004Saccharomyces cerevisiaeOther cell lines
Cardoso 2021 BEC MgG2021Cardoso LHD, Doerrier C, Gnaiger E (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. Bioenerg Commun 2021.1. https://doi.org/10.26124/bec:2021-0001MouseHeart
Krumschnabel 2014 Methods Enzymol2014Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. https://doi.org/10.1016/B978-0-12-416618-9.00009-1MouseNervous system


MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry, Fluorometry