SUIT-009

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SUIT-009

Description

SUIT9


Reference: A

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 Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-06-06)

Specific DLP protocols

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

SUIT-009

SUIT9 SUIT9
  • SUIT-009 AmR mt D021 simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate
SUIT-009 SUIT-9
  • 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

SUIT9 SUIT9
  • 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-009SUIT9
  • 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
MitoPedia: SUIT

Steps and respiratory states

1S;2D;3P;4Rot;5Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
2D SP S CII 1S;2D
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
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 entry into Q-junction Comment
## Tm TmE CIV CIV
## Azd ROX


Questions.jpg


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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

 YearReferenceOrganismTissue;cell
MiPNet20.14 AmplexRed H2O2-production2018-11-26
O2k-Protocols
O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed.
MouseHeart
Komlodi 2018 J Bioenerg Biomembr2018Komlódi T, Geibl FF, Sassani M, Ambrus A, Tretter L (2018) Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria. J Bioenerg Biomembr 10.1007/s10863-018-9766-8.Guinea pigHeart
Nervous system
Komlodi 2018 Methods Mol Biol2018Komlodi 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 Biomolecules2015
O2k-Protocols contents
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. Biomolecules 5:1319-38.
Human
Mouse
Heart
Nervous system
HEK
Krumschnabel 2015 Methods Mol Biol2015
O2k-Protocols contents
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.
MouseNervous system


MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


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