- high-resolution terminology - matching measurements at high-resolution
SUIT-026 AmR mt D064
Description
Abbreviation: Evaluation of ROS production by RET in isolated mitochondria
Reference: A - SUIT-026
SUIT number: D062_1S;2Rot;3D;4Ama
O2k-Application: AmR
- SUIT protocol pattern: 1S;2Rot;3D;4Ama
Communicated by Iglesias-Gonzalez J and Gnaiger E (last update 2019-06-26)
Steps and respiratory states
Step
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State
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Pathway
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Q-junction
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Comment - Events (E) and Marks (M)
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0DTPA
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|
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- 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.
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0SOD
|
|
|
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- SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
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0HRP
|
|
|
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- HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
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0AmR
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|
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|
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Step
|
State
|
Pathway
|
Q-junction
|
Comment - Events (E) and Marks (M)
|
1S
|
SL(n)
|
S
|
CII
|
1S
|
2Rot
|
SL(n)
|
S
|
CII
|
1S;2Rot
|
3D
|
SP
|
S
|
CII
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1S;2Rot;3D
|
4Ama
|
ROX
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|
|
1S;2Rot;3D;4Ama
- 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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- Coupling control
- » Coupling control state
- » ET capacity
- » OXPHOS capacity
- » LEAK respiration
- Pathway control
- » Electron transfer pathway
- » Fatty acid oxidation pathway control state, F
- » NADH electron transfer-pathway state, N
- » Succinate pathway control state, S
- » NS-pathway control state, NS
- » Glycerophosphate pathway control state, Gp
- » Complex IV single step, CIV
- » Anaplerotic pathway control state
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Glossary
- » List of SUIT states
- » SUIT concept
Strengths and limitations
Compare SUIT protocols
References
MitoPedia concepts:
SUIT protocol,
SUIT B,
Find
MitoPedia methods:
Fluorometry