SUIT-006 O2 mt D022

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SUIT-006 O2 mt D022

Description

1SRot;2D;2c;3(Omy);4U;5Ama.png

Abbreviation: CCP mt S(Rot)

Reference: A - SUIT-006 - Protocol for coupling control assessment in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)

SUIT number: D022_1SRot;2D;2c;(3Omy);4U;5Ama

O2k-Application: O2

SUIT protocol pattern: 1SRot;2D;2c;(3Omy);4U;5Ama

SUIT-006 O2 mt D022 is a coupling control protocol for mitochondrial preparations (tissue homogenate, isolated mitochondria and permeabilized cells). Different coupling control states are assessed (L(n) - P - E) at succinate pathway control state. The use of rotenone, that inhibits complex I, provides a specific coupling control protocol for S-pathway, avoiding reverse electron transfer (RET).

NOTE: In the DatLab software, the DLP file for SUIT-006 O2 mt D022 is provided without the addition of oligomycin. For using this protocol with oligomycin addition, a DLPU can be created, with the Omy event and mark. Using oligomycin in the preliminary assays with a not yet studied model is useful to determine whether in this kind of sample LEAK respiration in the absence of ADP (1SRot) may be overestimated due to the presence of residual and endogenous adenylates.

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


Representative traces

SUIT-006 O2 mt D022 traces.png

MitoPedia: SUIT

Steps and respiratory states

1SRot;2D;2c;3(Omy);4U;5Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1SRot SL S CII 1SRot
2D SP S CII 1SRot;2D
2c ScP S CII 1SRot;2D;2c
  • Succinate, S ( type S-pathway to Q).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
  • Addition of cytochrome c yields a test for integrity of the mtOM. 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 (P, OXPHOS-capacity with saturating [ADP]).
(3Omy) ScL(Omy) 1SRot;2D;2c;(3Omy)
  • Omy addition is skipped in SUIT-006 O2 mt D022
  • Succinate, S ( type S-pathway to Q).
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
4U SE S CII 1SRot;2D;2c;4U
5Ama ROX 1SRot;2D;2c;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


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Strengths and limitations

+ The combination of succinate with rotenone, that inhibits complex I, provides the best coupling control protocol specific for S-pathway, avoiding reverse electron transfer (RET).
+ Reasonable duration of the experiment.
+ This protocol can be extended with the Complex IV module.
- The DLP for SUIT-006 O2 mt D022 is provided with the oligomycin step skipped. However, this is a good control to analyse LEAK respiration. In samples that may contain endogenous adenylates and active ATPases, L(n) respiration may be overestimated. Therefore, in these conditions, L(Omy) is a better estimative of LEAK respiration. A DLPU can be created with the addition of oligomycin. It must be taken in consideration that high concentrations of oligomycin may affect the analysis of ET capacity, therefore the concentration applied must be previously tested.
- 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.


Compare SUIT protocols

  • SUIT-006: Extended protocol with SUIT-006 as the initial coupling control module.
  • SUIT-003: Coupling control protocol for non-permeabilized cells (ce), counterpart to SUIT-006 for mt-preparations (mtprep).

References

 YearReferenceOrganismTissue;cell
Krumschnabel 2014 Methods Enzymol2014
O2k-Protocols contents
Krumschnabel G, Eigentler A, Fasching M, Gnaiger E (2014) Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry. Methods Enzymol 542:163-81.
MouseNervous system


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry