SUIT-009

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SUIT-009

Description

Abbreviation: NS(P)

Reference: A  »Versions

SUIT-category: NS(P)

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

DLP applications

• SUIT-009 AmR mt D021
• SUIT-009 AmR pce D019
• SUIT-009 O2 mt D015
• SUIT-009 O2 pce D016

Steps and respiratory states

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.
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

References

Chang 2018 Life Sciences Meeting 2018 Innsbruck AT

Strengths and limitations

• 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.

+ Contribution of N pathway to the NS can be checked after the addition of Rot.

+ Reasonable duration of the experiment.

- Rox may be lower in substrate states earlier in the SUIT protocol. Therefore, this Rox measurement is frequently taken as a methodological control rather than as the final basis of Rox correction of mitochondrial respiration (mt).

- Careful washing is required after the experiment to avoid carry-over of inhibitors.

- CIV activity cannot be measured together with the fluorescence.

Compare SUIT protocols

MitoPedia concepts: MiP concept, SUIT protocol 

MitoPedia methods: Respirometry, Fluorometry