SUIT-009 O2 ce-pce D016

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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

SUIT-category: NS(P)

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

Originially 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 forSUIT-009 AmR pce D019 without using the Amplex UltraRed assay in order to detect the effect of the fluorescence 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)

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

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	SL(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	SP	S	CII	1S;2D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
2c	ScP	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	NSP	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	SP	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	AsTmE	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

• It is a protocol to investigate 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.

MitoPedia concepts: SUIT protocol, SUIT B, Find 

MitoPedia methods: Respirometry