SUIT-018

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SUIT-018

Description

SUIT-018

Abbreviation: O2 dependence-H2O2

Reference: A: A protocol for oxygen dependence of O2 flux and H2O2 flux in isolated mitochondria,tissue homogenate or permeabilized cells

SUIT protocol pattern: 1GMS;2D;-

SUIT-018 is a short protocol to study simultaneously the oxygen dependence of O2 flux and H2O2 flux in isolated mitochondria, tissue homogenate (except of liver homogenate) or permeabilized cells. In our experiments H2O2 flux shows linear dependence on oxygen concentration in LEAK, OXPHOS, ET states and in ROX state measured in MiR05-Kit, therefore, we recommend varying the oxygen level in the O2k-Chamber several times during the experiment. It is advisable to detect H2O2 flux in the same state either at high oxygen concentration (tissue hyperoxia; [O2]> ~100-110 µM) and at low oxyen concentration (tissue normoxia; [O2]< ~70-80 µM), or keep always the oxygen level in tisse normoxia. In this SUIT protocol several DLPs are listed to evaluate H2O2 flux in the NS-pathway in the LEAK and OXPHOS states. To decrease or increse the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.

Communicated by Komlodi T, Gnaiger E (last update 2020-06-09)

Specific SUIT protocols

SUIT-018 AmR mt D031

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

  • SUIT-018 AmR mt D031 simultaneous determination of O2 and H2O2 flux in isolated mitochondria, tissue homogenate and permeabilized cells at low oxygen concentration (tissue normoxia).

SUIT-018 AmR mt D040

SUIT-018 SUIT-018 AmR mt D040 O2 trace.png SUIT-018 AmR mt D040 AmR trace.png

  • SUIT-018 AmR mt D040 simultaneous determination of O2 and H2O2 flux in isolated mitochondria, tissue homogenate and permeabilized cells at various oxygen levels.

SUIT-018 AmR mt D041

SUIT-018 SUIT-018 AmR mt D041-DL-File O2.png SUIT-018 AmR mt D041-DL-File.png

  • SUIT-018 AmR mt D041 simultaneous determination of O2 and H2O2 flux in isolated mitochondria, tissue homogenate and permeabilized cells changing the oxygen concentration in the same protocol.

SUIT-018 O2 mt D054

SUIT-018

SUIT-018 O2 mt D054.png

  • SUIT-018 O2 mt D054 to measure O2 flux in isolated mitochondria, tissue homogenate and permeabilized cells as a control to test the effect of the fluorescence dye on the respiration.

SUIT-018 AmR ce-pce D068

Ce1;1Dig;2GMS;3D;4Ama.png

SUIT-018 AmR ce-pce D068 O2 trace.png SUIT-018 AmR ce-pce D068 AmR trace.png

  • SUIT-018 AmR ce-pce D068 simultaneous determination of O2 and H2O2 flux in permeabilized cells changing the oxygen concentration in the same protocol with testing the effect of the fluorescence dye on the ROUTINE-respiration.
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

  • Many fluorescence dyes can inhibit components of the ET system and thus, affecting the respiration. Therefore, a control run of the protocol should be done in the absence of the fluorescence dye.
+ Short duration of the experiment.
+ Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS state.
- 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-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential on the N-control state on isolated mitochondria and tissue homogenate.
  • SUIT-009 to investigate H2O2 production driven by the reverse electron transfer (RET) on isolated mitochondria, tissue homogenate and permeabilized cells.

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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