SUIT-026 O2 ce-pce D088

high-resolution terminology - matching measurements at high-resolution

SUIT-026 O2 ce-pce D088

Description

Abbreviation: RET (respiratory control) of SUIT-026 AmR ce-pce D087

Reference: B - SUIT-026 - Protocol for the respiratory control of SUIT-026 AmR ce-pce D087

SUIT number: D088_ce1;1Dig;1S;2Rot;3D;3c;4Ama

O2k-Application: O2

SUIT-026 O2 ce-pce D088 has been designed to serve as a respiratory control of the SUIT-026 AmR ce-pce D087 (protocol to study the RET-initiated ROS production in permeabilized cells (adding living cells into the chamber and permeabilizing the plasma membrane with digitonin titration.

Communicated by Cardoso LHD and Timon-Gomez A (last update 2024-06-10)

Representative traces

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	REN			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).
2Rot	S_L(n)	S	CII	1S;2Rot 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). Succinate pathway control state (S-pathway) after inhibiting CI with rotenone, which also inhibits the F-pathway.
3D	S_P	S	CII	1S;2Rot;3D Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
3c	S_P	S	CII	1S;2Rot;3D;3c 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]). Succinate, S ( type S-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Ama	ROX			1S;2Rot;3D;4Ama 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 serves as a respiratory control (since fluorescence dyes may inhibit components of the ET system) and quality control for the sample for SUIT-026 AmR ce-pce D088.

+ Short protocol.

+ The addition of cytochrome c to assess the mt outer membrane integrity provides a quality control of the sample, which cannot be performed in the protocol with Amplex UltraRed.

- This protocol does not provide information about all the coupling control states (LEAK, OXPHOS and ET). However, it is possible to create a DLPU by additing an Event&Mark for the uncoupler titration (4U) after ADP (3D) to obtain ET-state.

Compare SUIT protocols

SUIT-026 AmR ce-pce D087: Protocol to evaluate the RET production of ROS.
SUIT-006 O2 mt D022: CCP mtprep for S-pathway.

Chemicals and syringes

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

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

References

MitoPedia concepts: SUIT protocol, SUIT B, Find

MitoPedia methods: Fluorometry