SUIT-013 AmR ce D023

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SUIT-013 AmR ce D023

Description

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 intact cells. SUIT-013 AmR ce D023 is suitable for demonstration of the oxygen dependence in baker´s yeast. For further details see: MiPNet18.06 Amplex-Yeast. 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 and Iglesias-Gonzalez J  (last update 2019-06-06)
MitoPedia: SUIT

Steps and respiratory states

SUIT013 AmR ce D023.png


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)
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

References

 YearReferenceOrganismTissue;cell
MiPNet20.14 AmplexRed H2O2-production2018-11-26
O2k-Protocols
O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed.
MouseHeart
MiPNet18.06 Amplex-Yeast2018-11-25
O2k-Protocols
O2k-FluoRespirometry: Amplex UltraRed using freeze-dried baker’s yeast.
Saccharomyces cerevisiae
Fungi
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 Biomolecules2015
O2k-Protocols contents
Makrecka-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 Biol2015
O2k-Protocols contents
Krumschnabel 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


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry, Fluorometry