SUIT-018 AmR ce-pce D068

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SUIT-018 AmR ce-pce D068

Description

Ce1;1Dig;1GMS;2D;3Ama.png

Abbreviation: O2 dependence-H2O2

Reference: A -SUIT-018

SUIT number: D068_ce1;1Dig;1GMS;2D;3Ama

O2k-Application: AmR

MitoPedia: SUIT - SUIT protocol for simultaneous determination of O2 and H2O2 flux in permeabilized cells at various oxygen concentrations
SUIT protocol pattern: ce1;1Dig;1GMS;2D-

SUIT-018 AmR ce-pce D068 is a protocol to simultaneously study the oxygen dependence of O2 flux and H2O2 flux in permeabilized cells, and to test the chemical inhibitory effect of the AmR assay (SOD, HRP and AmR) on ROUTINE-respiration of living cells. The oxygen concentration in the chamber can be decreased by injecting nitrogen with a syringe. To test the inhibitory effect of the AmR assay, DTPA, SOD, HRP and AmR are added in ROUTINE. However, the inhibitory effect of the AmR assay can be tested in separate protocols: SUIT-003 AmR ce D058 and SUIT-003 AmR ce D059. For further information, see: Amplex UltraRed page. To decrease or increase the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.

Communicated by  Komlodi T, Cecatto C and Gnaiger E (last update 2020-06-09)

Representative traces

SUIT-018 AmR ce-pce D068 O2 trace.png

SUIT-018 AmR ce-pce D068 AmR trace.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1GMS;2D;3Ama.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.
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)
1Dig ROX ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
1GMS GMSL(n) NS CI&CII ce1;1Dig;1GMS
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • Non-phosphorylating resting state (LEAK state); LEAK respiration, L(n), in the absence of ADP, ATP, AMP (no adenylates).
2D GMSP NS CI&CII ce1;1Dig,1GMS;2D
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
3Ama ROX ce1;1Dig,1GMS;2D;3Ama
  • 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

+ Simple protocol to evaluate the oxygen dependence of H2O2 flux in LEAK and OXPHOS.
+ Inhibitory effect of the AmR assay can be tested in the same protocol on ROUTINE respiration.
- Reoxygenation and nitrogen injection can lead to bubble formation in the O2k-Chamber.
- CIV activity and cytochrome c test cannot be performed together with the fluorescence.

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References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Fluorometry