SUIT-018 AmR mt D031

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SUIT-018 AmR mt D031

Description

1GMS;2D;3Ama.png

Abbreviation: NS(GM)

Reference: A: for simultaneous determination of O2 and H2O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) at low oxygen concentration (tissue normoxia)-SUIT-018

SUIT number: D031_1GMS;2D;3Ama

O2k-Application: AmR

MitoPedia: SUIT
SUIT-category: NS(GM)
SUIT protocol pattern: linear 1GMS;2D-

SUIT-018 AmR mt D031 is a short protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 production on mitochondrial preparations. Is it possible to run the protocol under different oxygen concentration conditions: 1.) under tissue normoxia (50-60 µM = [O2]), 2.) under tissue hyperoxia (> 120 µM = [O2]) or 3.) you can change the oxygen concentration during the experiment. The oxygen concentration in the chamber can be altered injecting nitrogen with a syringe. We recommend to run SUIT-018 AmR mt D040 simultaneously at high oxygen concentration as a control.

Communicated by  Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-04-03)
MitoPedia: SUIT

Steps and respiratory states

SUIT-018

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
0DTPA
  • DTPA is an iron chelator, which decreases the chemical fluorescence background created by the Amplex UltraRed assay. Administration of DTPA into the O2k-chamber is recommended before all other chemicals because the iron chelation capacity of the compound is time-dependent (approx. 10-15 min). However, the experiments can be carried out in the absence of DTPA.
0SOD
  • SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
0HRP
  • HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
0AmR
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1GMS GMSL(n) NS CI&CII 1GMS
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(n), in the absence of ADP, ATP, AMP (no adenylates).
2D GMSP NS CI&CII 1GMS;2D
  • 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.
3Ama ROX 1GMS;2D;3Ama
  • 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).

Strengths and limitations

+ Reasonable duration of the experiment.
+ Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS.
- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
- Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber.
- CIV activity and cytochrome c test cannot be performed together with the fluorescence.

Compare SUIT protocols

  • SUIT-018 AmR mt D041 for simultaneous determination of O2 and H2O2 flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) changing the oxygen concentration in the same protocol.
  • SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential on the N-control state in mitochondrial preparations.
  • SUIT-009 to investigate H2O2 production driven by the reverse electron transfer (RET) in isolated mitochondria, tissue homogenate and permeabilized cells.

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 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
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
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



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