SUIT-003 AmR ce D017

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SUIT-003 AmR ce D017

Description

Ce1;ce2P;ce3Omy;ce4U;ce5Rot;ce6S;ce7Ama.png

Abbreviation: CCP-ce S permeability test

Reference: B: CCP(P) and cell membrane permeability test with succinate - SUIT-003

SUIT number: D017_ce1;ce2P;ce3Omy;ce4U;ce5Rot;ce6S;ce7Ama

O2k-Application: AmR

MitoPedia: SUIT - coupling control protocol (CCP) with pyruvate and cell membrane permeability test
SUIT protocol pattern: ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama

This protocol has been designed to study the H2O2 production in various coupling control states and test the cell membrane permeability with the same protocol using living cells. Pyruvate is added to support the ROUTINE respiration of living cells when they are in a respiratory for mitochondrial preparations (i.e. MiR05). To initiate LEAK-respiration oligomycin is added, inhibiting the ATP-synthase and leading to an increased in the H2O2 production. Whereas titrations of uncoupler results in a decreased H2O2 generation. However, one of the main problems with living cells is that the H2O2 flux is very low or hardly detectable due to the scavenger activity of the antioxidant enzymes in the cytosol.

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, Doerrier C, Iglesias-Gonzalez J and Cardoso LH (last update 2019-07-05)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2P;ce3Omy;ce4U;ce5Rot;ce6S;ce7Ama.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.
ce1P ROUTINE ce1;ce1P; ROUTINE, R
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce2Omy L(Omy) ce1;ce1P;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U E ce1;ce1P;ce2Omy;ce3U
ce4Rot ROX ce1;ce1P;ce2Omy;ce3U;ce4Rot
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
ce5S ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S
ce6Ama ROX ce1;ce1P;ce2Omy;ce3U;ce4Rot;ce5S;ce6Ama
  • 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

  • When MiR05 is used, the addition of pyruvate is recommended to provide an external fuel substrate.
  • When culture medium (e.g. DMEM) is used, application of phenol red is not advisable, since it can interfere with the fluorescence signal.
+ Reasonable duration of the experiments.
- Minor changes in the H2O2 flux can be seen with living cells because of the action of the antioxidant enzymes in the cytosol, which scavenge the H2O2 produced. (AmR is not permeable to the cell membrane, therefore, only the H2O2 that crosses the plasma membrane can be measured)

Compare SUIT protocols

  • SUIT-013 AmR ce D023: SUIT-013 has been designed to study the oxygen dependence of H2O2 production in intact cells.

References

 YearReferenceOrganismTissue;cell
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 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 B, Find 


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