SUIT-006 O2 mt D022

high-resolution terminology - matching measurements at high-resolution

SUIT-006 O2 mt D022

Description

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

O2k-Application: O2

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, Doerrier C and Gnaiger E (last update 2024-09-19)

Representative traces

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1SRot	S_L	S	CII	1SRot Succinate, S ( type S-pathway to Q). Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway. Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	S_P	S	CII	1SRot;2D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	Sc_P	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 (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)	Sc_L(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	S_E	S	CII	1SRot;2D;2c;4U Succinate, S ( type S-pathway to Q). Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
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).

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Coupling control

» Coupling control state

» ET capacity

» OXPHOS capacity

» LEAK respiration

Pathway control

» Electron transfer pathway

» Fatty acid oxidation pathway control state, F

» NADH electron transfer-pathway state, N

» Succinate pathway control state, S

» NS-pathway control state, NS

» Glycerophosphate pathway control state, Gp

» Complex IV single step, CIV

» Anaplerotic pathway control state

Main fuel substrates

» Glutamate, G

» Glycerophosphate, Gp

» Malate, M

» Octanoylcarnitine, Oct

» Pyruvate, P

» Succinate, S

Glossary

» List of SUIT states

» SUIT concept

Strengths and limitations

This protocol is recommended when the aim is to analyse coupling control in mtprep (see MitoPedia: Sample preparations) at Succinate pathway control state.

+ 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).

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1SRot	Succinate (S) and Rotenone (Rot)
2D	ADP (D)
2c	Cytochrome c (c)
(3Omy)	Oligomycin (Omy)	This step can be skipped
4U	Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U)	Can be substituted for other uncoupler
5Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

References

	Year	Reference	Organism	Tissue;cell
Krumschnabel 2014 Methods Enzymol	2014	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. https://doi.org/10.1016/B978-0-12-416618-9.00009-1	Mouse	Nervous system

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Respirometry