SUIT-018 AmR mt D031

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SUIT-018 AmR mt D031

Description

1GMS;2D;3Ama.png

Abbreviation: O2 dependence-H2O2

Reference: A -SUIT-018 - SUIT protocol 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 number: D031_1GMS;2D;3Ama

O2k-Application: AmR


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. To decrease or increase the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration. We recommend to run SUIT-018 AmR mt D040 simultaneously at high oxygen concentration as a control.

Communicated by  Komlodi T, Gnaiger E (last update 2019-09-24)

Representative traces

SUIT-018 mt AmR D031 O2 DL-Files.png SUIT-018 AmR mt D031 DL-Files.png

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.

Chemicals and syringes

Step Chemical(s) and link(s) Comments
H2O2 Hydrogen Peroxide (H2O2)
Step Chemical(s) and link(s) Comments
0DTPA DTPA This step can be skipped
0SOD Superoxide Dismutase (SOD)
0HRP Horseradish peroxidase (HRP)
0AmR Amplex UltraRed (AmR)
Step Chemical(s) and link(s) Comments
1GMS Glutamate (G), Malate (M) and Succinate (S)
2D ADP (D)
3Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.

References

 YearReferenceOrganismTissue;cell
Komlodi 2021 MitoFit AmR-O22021-11-08Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. MitoFit Preprints 2021.10. doi:10.26124/mitofit:2021-0010Saccharomyces cerevisiaeOther cell lines
MiPNet24.10 H2O2 flux analysis2021-10-22
O2k-Protocols
Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
MiPNet20.14 AmplexRed H2O2-production2019-06-24
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
Makrecka-Kuka 2015 Biomolecules2015Makrecka-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 Biol2015Krumschnabel 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



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