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SUIT-006 AmR mt D048

From Bioblast

high-resolution terminology - matching measurements at high-resolution

SUIT-006 AmR mt D048



Abbreviation: CCP mt PM - AmR

Reference: A: short protocol for simultaneous determination of O2 flux and H2O2 in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells)- SUIT-006

SUIT number: D048_1PM;2D;3Omy;4U;5Ama

O2k-Application: AmR

SUIT-006 AmR mt D048 is a short protocol to study simultaneously the O2 flux and H2O2 flux (with the fluorescent dye Amplex UltraRed), in particular to study the dependence of H2O2 flux on the mt-membrane potential. In this protocol , the NADH Electron transfer-pathway state can be assessed in mitochondrial preparations such as isolated mitochondria, tissue homogenates (except of liver homogenate) and permeabilized cells (already permeabilized when they are added to the chamber) in a wide variety of species and tissues. After addition of the NADH-linked substrates, the addition of ADP depolarises the mt-membrane which leads to the decrease of the H2O2 flux. Oligomycin (Omy) is used to induce a LEAK state of respiration via the inhibition of the ATP synthase. Omy hyperpolarises the mt-membrane resulting in increased H2O2 production, while the addition of the uncoupler decreases the H2O2 generation owing to the depolarisation of the mt-membrane. Since higher concentrations of Omy can decrease the ET state induced upon addition of uncoupler, the optimal concentration of Omy has to be determined.

According to our results, the H2O2 production is linearly dependent on the O2 concentration in MiR05-Kit, therefore, during the measurements the O2 concentration has to be well-defined. In this protocol, we suggest not to go under ~100 µM O2. SUIT-018 AmR mt D041 has been designed to study O2 dependence of H2O2 flux in mitochondrial preparations.

Communicated by  Komlodi T, Cardoso LHD, Gnaiger E (last update 2019-07-05) 

Representative traces

SUIT-006 AmR mt D048 O2.png SUIT-006 AmR mt D048.png

MitoPedia: SUIT

Steps and respiratory states


Step State Pathway Q-junction Comment - Events (E) and Marks (M)
  • 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.
  • SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
  • HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1PM PML(n) 1PM
3Omy PML(Omy) 1PM;2D;3Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin is optional depending on the experimental question to resolve. If it is known that Ln has the same values for LOmy for a kind of sample and substrates, this step may be skipped. If the carrier of oligomycin (EtOH) is used instead, it is possible to evaluate whether it affects OXPHOS. In these cases, LEAK state is not reached again.
4U PME 1PM;2D;3Omy;4U
5Ama ROX 1PM;2D;3Omy;4U;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).


Click to expand or collaps

Strengths and limitations

  • This protocol can be runned under low oxygen concentration to test the oxygen dependence of mt-ROS production. For more technical details see: SUIT-018 AmR mt D031, SUIT-018 AmR mt D041.
  • 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.
- CIV activity cannot be determined and cytochrome c test cannot be performed together with the fluorescence dyes.
+ This protocol can be extended with the Complex IV module in the following protocols: SUIT-006 O2 mt D047.
+ Cytochrome c test can be performed in the following protocols:SUIT-006 O2 mt D047.
- This protocol cannot be runned with liver homogenate.
- Omy concentration has to be determined. Higher concentrations of Omy may inhibit the ET state.

Compare SUIT protocols

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
1PM Pyruvate (P) and Malate (M)
2D ADP (D)
3Omy Oligomycin (Omy)
4U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler
5Ama Antimycin A (Ama)
Suggested stock concentrations are shown in the specific DL-Protocol.


MiPNet24.10 H2O2 flux analysis2021-10-22
Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
Komlodi 2021 BEC AmR-O22021Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. cerevisiaeOther cell lines
MiPNet20.14 AmplexRed H2O2-production2019-06-24
O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed.
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
Skeletal muscle
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
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.
Nervous system

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Fluorometry