SUIT-009

high-resolution terminology - matching measurements at high-resolution

SUIT-009

Description

Reference: B

SUIT protocol pattern:1S;2D;3P;4Rot-

SUIT-009 is a short protocol for simultaneous determination of O₂ flux and the rate of H₂O₂ production (H₂O₂ flux) on isolated mitochondria, tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET)-related H₂O₂ production in the LEAK state. Pyruvate (P) promotesNADH-pathway (N), and usually in the presence of ADP, H₂O₂ flux is not increased further. Antimycin A (Ama) inhibits Complex III (CIII) and could increase the H₂O₂ flux. The sensitivity of the Amplex UltraRed® assay (for determining H₂O₂ production) changes over the experimental time and upon addition of chemicals. To correct H₂O₂ flux for the sensitivity changes several H₂O₂ calibration steps are done during the experiment. The measurement is carried out in MiR05-Kit at 37 °C. For O₂-Application, apply SUIT-009 O2 mt D015 or SUIT-009 O2 ce-pce D016; for O₂ and H₂O₂ measurements (AmR), apply SUIT-009 AmR mt D021 or SUIT-009 AmR ce-pce D019.

Communicated by Komlodi T, Gnaiger E (last update 2024-09-19)

Specific DLP protocols

SUIT-009 AmR mt D021 and SUIT-009 O2 mt D015

SUIT-009 AmR mt D021 simultaneous determination of O₂ and H₂O₂ flux on isolated mitochondria and tissue homogenate

SUIT-009 O2 mt D015 determination of O₂ flux on isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber) as a control for SUIT-009 AmR mt D021

SUIT-009 AmR ce-pce D019 and SUIT-009 O2 ce-pce D016

SUIT-009 AmR ce-pce D019 simultaneous determination of O₂ and H₂O₂ flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin )

SUIT-009 O2 ce-pce D016 determination of O₂ flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin) as a control for SUIT-009 AmR ce-pce D019

Steps and respiratory states

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.
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

This protocol allows to test:

any Complex I defect, which would lead to a decrease in the N-linked respiration and a reduced reverse electron transfer-supported ROS production.
any Complex II defect, which would result in decreased S-linked respiration and ROS production.

A succinate concentration > 10 mM might be required to reach saturating S-OXPHOS capacity.
This protocol can be run under low oxygen concentration to test the oxygen dependency of mt-ROS production. For more technical details, see: SUIT-018 AmR mt D031, SUIT-018 AmR mt D041.
In some cell lines e.g. HEK293T, pyruvate does not have any effect because of the downregulation of the N-linked pathway.
To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: SUIT 003 AmR ce D058 with AmR and SUIT 003 AmR ce D059 with carrier titration as a control. These two protocols have to be done in parallel.

+ Reasonable duration of the experiment.

+ This protocol can be extended with the Complex IV module in the following protocols: SUIT-009 O2 mt D015 and SUIT-009 O2 ce-pce D016.

+ Cytochrome c test can be performed in the following protocols: SUIT-009 O2 mt D015 and SUIT-009 O2 ce-pce D016, but not simultaneously with the fluorescence assay.

Compare SUIT protocols

SUIT-006 AmR mt D048 to investigate the dependence of H₂O₂ flux on the mt-membrane potantial on the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
SUIT-018 is a short protocol to study oxygen dependence of O₂ flux and H₂O₂ production on isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).

References

	Year	Reference	Organism	Tissue;cell
MiPNet24.10 H2O2 flux analysis	2021-10-22	Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
Komlodi 2021 BEC AmR-O2	2021	Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004	Saccharomyces cerevisiae	Other cell lines
MiPNet20.14 AmplexRed H2O2-production	2019-06-24	O2k-FluoRespirometry: HRR and simultaneous determination of H₂O₂ production with Amplex UltraRed.	Mouse	Heart
Komlodi 2018 Methods Mol Biol	2018	Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55.	Human Mouse	Skeletal muscle HEK
Makrecka-Kuka 2015 Biomolecules	2015	Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. https://doi.org/10.3390/biom5031319	Human Mouse	Heart Nervous system HEK
Krumschnabel 2015 Methods Mol Biol	2015	Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. https://doi.org/10.1007/978-1-4939-2257-4_22	Mouse	Nervous system

MitoPedia concepts: MiP concept, SUIT B

MitoPedia methods: Fluorometry