SUIT-009 O2 ce-pce D016

high-resolution terminology - matching measurements at high-resolution

SUIT-009 O2 ce-pce D016

Description

Abbreviation: NS(P)

Reference: B: SUIT-009 short protocol for mt-respiration in pce.

SUIT number: D016_ce1;1Dig;1S;2D;3P;4Rot;5Ama

MitoPedia: SUIT - SUIT reference protocol

SUIT-category: NS(P)

SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot;5Ama

This is a short protocol for simultaneous determination of O₂ flux and the rate of H₂O₂ production (H₂O₂ flux) on permeabilized cells. It is used as a control experiment for SUIT-009 AmR pce D019 without using the Amplex UltraRed assay in order to detect the effect of the fluorescent dye on the O₂ flux. We do not recommend this protocol for determination of the O₂ flux in the absence of Rotenone. For O2-Application with isolated mitochondria and tissue homogenate, choose SUIT-009 O2 mt D015; for O2 and H₂O₂ measurements (AmR) with permeabilized cells, choose SUIT-009 AmR pce D019.

Communicated by Iglesias-Gonzalez J,  Komlodi T and Gnaiger E (last update 2019-02-12)

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
ce1	ROUTINE			ce1 ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
1Dig	Dig			ce1;1Dig Optimum effective digitonin concentration for complete plasma membrane permeabilization.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1S	S_L(n)	S	CII	1S Succinate, S ( type S-pathway to Q). Non-phosphorylating resting state (LEAK state); LEAK respiration L(n) in the absence of ADP, ATP, AMP (no adenylates).
2D	S_P	S	CII	1S;2D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	Sc_P	S	CII	1S;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]).
3P	NS_P	NS	CI&II	1S;2D;3P Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function. OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Rot	S_P	S	CII	1S;2D;3P;4Rot Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state. Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
5Ama	ROX			1S;2D;3P;4Rot;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).

Step	Respiratory state	Pathway control	ET-Complex	Comment
## AsTm	AsTm_E	CIV	CIV
## Azd	CHB

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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

We do not recommend this protocol for respiration, since rotenone is not added at the beginning to avoid oxaloacetate inhibition towards succinate dehydrogenase. This protocol can be used as a control to investigate the effect of the fluorescence dyes on the respiration.
It is a protocol to analyse the S- and NS-pathway linked respiration.
For H₂O₂-Application, choose SUIT-009 AmR pce D019.

+ Cytochrome c test can be performed in the S-OXPHOS state.

+ Reasonable duration of the experiment.

+ This protocol can be extended with the Complex IV module.

- A succinate concentration > 10 mM might be required to reach saturating S-OXPHOS capacity.

- In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.

Compare SUIT protocols

SUIT-006 O2 mt D022: to investigate the S(Rot)-linked respiration.

References

	Year	Reference	Organism	Tissue;cell
Chang 2018 Life Sciences Meeting 2018 Innsbruck AT	2018	pH dependence of mitochondrial respiration and H₂O₂ production in oral cancer cells – a pilot study.	Human	HEK Kidney

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Respirometry