SUIT-003 AmR ce D059

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SUIT-003 AmR ce D059

Description

Ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama.png

Abbreviation: Amr effect on ce - control

Reference: B: Experimental control for SUIT-003 AmR ce D058

SUIT number: D059_ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama

O2k-Application: AmR

MitoPedia: SUIT - Carrier control protocol to SUIT-003 AmR ce D058
SUIT protocol pattern: ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama

SUIT-003 AmR ce D059 is a control protocol of SUIT-003 AmR ce D058 to study the effect of the carrier titrations on the respiration in a parallel experiment. This protocol should be run in parallel with SUIT-003 AmR ce D058 to compare the respiratory capacities and the Flux Control Ratios (FCR) in order to detect any inhibition of the respiratory capacities by the Amplex UltraRed or other components of the ROS assay.

Communicated by  Garcia-Souza LF, Komlodi T and Gnaiger E(last update 2019-06-26)


MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1H2O;ce1MiR05;ce1DMSO;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.
ce1H2O ROUTINE ce1;ce1H2O
  • SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
ce1MiR05 ROUTINE ce1;ce1H2O;ce1MiR05
  • HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
ce1DMSO ROUTINE ce1;ce1H2O;ce1MiR05;ce1DMSO
ce2Omy LOmy ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U E ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U
ce4Rot ROX ce1;ce1H2O;ce1MiR05;ce1DMSO;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;ce1H2O;ce1MiR05;ce1DMSO;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 effect of the carrier titrations on the respiration in parallel to SUIT-003 AmR ce D058. Therefore, instead of SOD, HRP and AmR, the carriers H2O; MiR05; and DMSO are titrated.
  • It is recommended to run this test with every different sample type used.
+ 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

MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry