SUIT-009 AmR ce-pce D019

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SUIT-009 AmR ce-pce D019

Description

SUIT9

Abbreviation: NS(P)

Reference: A: short protocol for H2O2 (AmR) in ce: non-permeabilized cells, pce: permeabilized cells -SUIT-009

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

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

SUIT-009 is a short protocol for simultaneous determination of O2 flux and the rate of H2O2 production (H2O2 flux) in permeabilized cells (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). Succinate (S) supports the reverse electron transfer (RET)-related H2O2 production in the LEAK state. 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) at the Qo level and could increase the H2O2 flux. The sensitivity of the Amplex UltraRed® assay (for determining H2O2 production) changes 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 experimental control for the O2-Application should be carried out with SUIT-009 O2 ce-pce D016.

Communicated by Huete-Ortega M, Cardoso LH, Doerrier C, Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2019-04-01)
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.
2Dig 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).



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Strengths and limitations

  • It is a protocol design to analyse:
1. the S- and NS-pathway linked respiration and ROS production.
2. any Complex I defect, which would lead to a decrease in the N-linked respiration and a reduced reverse electron transfer-supported ROS production.
3. any Complex II defect, which would result in an altered S-linked respiration and ROS production.
  • SUIT-009 O2 ce-pce D016 determination of O2 flux on permeabilized cells as a control for SUIT-009 AmR ce-pce D019 to test the inhibitory effect of the fluorescence dye on the mt-respiration.
+ This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which might be inhibited by ADP due to the depolarisation of the mt-membrane.
+ Short duration of the experiment.
- CIV activity and cytochrome c test cannot be performed together with the fluorescence assay.
- 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

  • SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potantial on the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
  • SUIT-018 is a short protocol to study oxygen dependence of O2 flux and H2O2 production on isolated mitochondria or tissue homogenate (except of liver).

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Fluorometry