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Difference between revisions of "SUIT-009 AmR ce-pce D019"

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== References ==
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Revision as of 13:03, 23 January 2019


high-resolution terminology - matching measurements at high-resolution


SUIT-009 AmR ce-pce D019

Description

SUIT9

Abbreviation: NS(P)

Reference: A: SUIT-009 short protocol for H2O2 (AmR) in pce.

SUIT number: D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama

SUIT-category: NS(P)
SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot;5Ama

SUIT 9 is a short protocol for simultaneous determination of O2 flux and the rate of H2O2 production (H2O2 flux) on isolated mitochondria,tissue homogenate (except of liver) and permeabilized cells. Succinate (S) supports the reverse electron transfer (RET) initiated H2O2 production in the LEAK state. Addition of ADP decreases H2O2 flux due to depolarization of the mitochondrial membrane. Pyruvate (P) supports NADH-pathway (N), and usually in the presence of ADP it does not increase further the H2O2 flux. Antimycin A (Ama) inhibits Complex III (CIII) and results in the elevation of the H2O2 flux. The sensitivity of the Amplex UltraRed® assay (for determining H2O2 production) is changing over the experimental time and upon addition of chemicals. To correct H2O2 flux for the sensitivity changes several H2O2 calibration steps are done during the experiment. The measurement is carried out in MiR05-Kit at 37 °C.

Communicated by Iglesias-Gonzalez J,  Komlodi T and Gnaiger E (last update 2019-01-22)

DLP applications

References

MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1S;2D;3P;4Rot;5Ama.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.
1Dig Dig ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
2D SP S CII 1S;2D
3P NSP NS CI&II 1S;2D;3P
4Rot SP S CII 1S;2D;3P;4Rot
5Ama ROX
  • 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).


Questions.jpg


Click to expand or collaps

Strengths and limitations

+ You can investigate in the absence of ADP the reverse electron transfer supported H2O2 production in S respiring mitochondria,which will be inhibited by ADP owing to the depolarisation of the mt-membrane.
- CIV activity and Cytochrome c test cannot be performed together with the fluorescence.
- The H2O2 flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of the cytosol.
- In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.


Compare SUIT protocols

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Fluorometry