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SUIT-013 AmR ce D023

From Bioblast

high-resolution terminology - matching measurements at high-resolution

SUIT-013 AmR ce D023


SUIT013 AmR ce D023.png

Abbreviation: O2 dependence of H2O2 production ce

Reference: A SUIT-013

SUIT number: D023_ce1

O2k-Application: AmR

MitoPedia: SUIT
SUIT protocol pattern: ce1

This protocol has been designed to study the oxygen dependence of H2O2 production in living cells. SUIT-013 AmR ce D023 is suitable for demonstration of the oxygen dependence in baker´s yeast. For further details see: Komlodi 2021 MitoFit AmR-O2. The respiration medium has to be chosen carefully, since the Amplex UltraRed assay leads to artefact formation with the components of the buffer.

Communicated by  Komlodi T (last update 2024-03-07)

Representative traces

SUIT-013 O2 D023.png SUIT-013 AmR D023.png

MitoPedia: SUIT

Steps and respiratory states

SUIT013 AmR ce D023.png

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)
ce1 ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.


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Strengths and limitations

  • 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.
+ Quick demonstration of the oxygen dependence of H2O2 production.
+ No externally added substrates are required with yeast.

Compare SUIT protocols


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
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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.
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MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry, Fluorometry