SUIT-009 O2 ce-pce D016

high-resolution terminology - matching measurements at high-resolution

SUIT-009 O2 ce-pce D016

Description

Reference: B - SUIT-009 - short protocol for mt-respiration in ce: non-permeabilized cells, pce: permeabilized cells.

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

SUIT-009 O2 ce-pce D016 is a short protocol to investigate O₂ flux in permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). It is recommended as a control experiment for SUIT-009 AmR ce-pce D019 without using the Amplex UltraRed assay in order to detect the effect of the fluorescence dye on the O₂ flux and with the cytochrome c to assess the integrity of the mitochondrial outer membrane. We do not recommend this protocol for determination of the O₂ flux in the absence of Rotenone. The following Coupling-control protocol can be used to assess Succinate-linked respiration with Rotenone: SUIT-006 O2 mt D022.

Communicated by Doerrier C, Huete-Ortega M, Komlodi T, Gnaiger E (last update 2019-06-06)

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

Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

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 to assess just respiration since rotenone is not added at the beginning and does not prevent the inhibitory effect of oxaloacetate on the 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 ce-pce D019.

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

+ Short duration of the experiment.

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

- A succinate concentration > 10 mM might be required to reach 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.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
1Dig	Digitonin (Dig)

Step	Chemical(s) and link(s)	Comments
1S	Succinate (S)
2D	ADP (D)
2c	Cytochrome c (c)
3P	Pyruvate (P)
4Rot	Rotenone (Rot)
5Ama	Antimycin A (Ama)

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

References

MitoPedia concepts: SUIT protocol, SUIT B, Find 

MitoPedia methods: Respirometry