SUIT-003 AmR ce D058

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SUIT-003 AmR ce D058

Description

Ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama.png

Abbreviation: AmR effect on ce

Reference: B: Coupling control in intact cells with oligomycin to study the inhibitory effect of the AmR assay components on the respiration ; ce: non-permeabilized cells -SUIT-003

SUIT number: D058_ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama

O2k-Application: AmR

MitoPedia: SUIT - Protocol to analyse AmR effect on living cells respiration
SUIT protocol pattern: ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama

SUIT-003 O2 ce D058 has been designed to analyze the possible inhibitory effect of the Amplex UltraRed assay on the living cells coupling control state. Usually, the Superoxide dismutase and the Horseradish peroxidase do not show any inhibitory effect over the respiration. However, the Amplex UltraRed seems to inhibit ROUTINE respiration and the ET-capacity at high concentrations. For this reason, the inhibitory effect of Amplex UltraRed should be tested with each type of sample used prior to the study of ROS production. SUIT-003 AmR ce D059 should be performed simultaneously as a carrier control titration.

Communicated by  Komlodi T, Garcia-Souza LF and Gnaiger E (last update 2019-06-24)


MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama.png

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.
ce1SOD ROUTINE ce1;ce1SOD
  • SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
ce1HRP ROUTINE ce1;ce1SOD;ce1HRP
  • HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
ce1AmR ROUTINE ce1;ce1SOD;ce1HRP;ce1AmR
ce2Omy LOmy ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U E ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U
ce4Rot ROX ce1;ce1SOD;ce1HRP;ce1AmR;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.
ce5Ama ROX ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U;ce4Rot;ce5Ama
  • 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).


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Strengths and limitations

  • This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay over living cells respiration.
  • It is recommended to run this test with every different sample type used.
  • A carrier control titration should be run in parallel (SUIT-003 AmR ce D059).
+ Reasonable duration of the experiment.
- The concentration of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

References

 YearReferenceOrganismTissue;cell
MiPNet24.10 H2O2 flux analysis2019-08-05
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
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
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

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry