SUIT-018 AmR mt D040

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SUIT-018 AmR mt D040

Description

SUIT-018

Abbreviation: NS(GM)

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

SUIT number: D040_1GMS;2D;3Ama

O2k-Application: AmR

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

SUIT-018 AmR mt D040 is a protocol to simultaneously study the oxygen dependence of O2 flux and H2O2 flux in mitochondrial preparations. In experiments, H2O2 flux shows linear dependence on oxygen concentration in LEAK, OXPHOS, ET states and in ROX state measured in MiR05-Kit medium. It is therefore recommended to vary the oxygen level in the O2k-Chamber several times during an experiment. It is also advisable to either detect H2O2 flux at the same state for high (tissue hyperoxia; [O2]> ~100-110 µM) and low (tissue normoxia; [O2]< ~70-80 µM) oxygen concentration, or keep within tissue normoxia. A further recommendation is to reduce the oxygen concentration in the O2k-Chamber before the sample addition. To decrease or increase the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.

Communicated by Tindle-Solomon L, Cecatto C, Komlodi T, Gnaiger E (last update 2020-06-25)


Representative traces

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

+ Simple protocol to evaluate the oxygen dependence of H2O2 production in LEAK and OXPHOS states.
+ Combination of N- and S-pathways is physiologically more relevant than using simple substrate to support a specific mitochondrial pathway.
+ Option at the end of the protocol to study the effect of oxygen concentration in the ROX state (in the presence of Ama) both on the O2 and H2O2 fluxes.
- It is advisable to adjust the optimal sample concentration in the O2k-Chamber beforehand to avoid unnecessary reoxygenations (high sample concentration) or very long protocols (low sample concentration causing slower oxygen consumption in the O2k-Chamber and thus, delays in reaching the required oxygen concentrations).
- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.
- Reoxygenation and nitrogen injection can 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 D041a simple protocol 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 a protocol to investigate the dependence of H2O2 flux on mt-membrane potential on the N-control state in mitochondrial preparations.
  • SUIT-009 a protocol 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 BEC AmR-O22021-12-21Komlódi 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:2021-0004Saccharomyces 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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