- SUIT protocol pattern:1S;2D;3P;4Rot-
SUIT-009 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)-related H2O2 production in the LEAK state. Pyruvate (P) promotesNADH-pathway (N), and usually in the presence of ADP, H2O2 flux is not increased further. Antimycin A (Ama) inhibits Complex III (CIII) 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 measurement is carried out in MiR05-Kit at 37 °C. For O2-Application, apply SUIT-009 O2 mt D015 or SUIT-009 O2 ce-pce D016; for O2 and H2O2 measurements (AmR), apply SUIT-009 AmR mt D021 or SUIT-009 AmR ce-pce D019.
Communicated by Iglesias-Gonzalez J, Komlodi T and Gnaiger E (last update 2020-06-12)
Specific DLP protocols
SUIT-009 AmR mt D021 and SUIT-009 O2 mt D015
- SUIT-009 AmR mt D021 simultaneous determination of O2 and H2O2 flux on isolated mitochondria and tissue homogenate
- SUIT-009 O2 mt D015 determination of O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber) as a control for SUIT-009 AmR mt D021
SUIT-009 AmR ce-pce D019 and SUIT-009 O2 ce-pce D016
- SUIT-009 AmR ce-pce D019 simultaneous determination of O2 and H2O2 flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin )
- SUIT-009 O2 ce-pce D016 determination of O2 flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin) as a control for SUIT-009 AmR ce-pce D019
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
|Step||Respiratory state||Pathway control||ET-Complex entry into Q-junction||Comment|
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- Pathway control
Strengths and limitations
- This protocol allows to test:
- any Complex I defect, which would lead to a decrease in the N-linked respiration and a reduced reverse electron transfer-supported ROS production.
- any Complex II defect, which would result in decreased S-linked respiration and ROS production.
- A succinate concentration > 10 mM might be required to reach saturating S-OXPHOS capacity.
- This protocol can be run under low oxygen concentration to test the oxygen dependency of mt-ROS production. For more technical details, see: SUIT-018 AmR mt D031, SUIT-018 AmR mt D041.
- In some cell lines e.g. HEK293T, pyruvate does not have any effect because of the downregulation of the N-linked pathway.
- To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: SUIT 003 AmR ce D058 with AmR and SUIT 003 AmR ce D059 with carrier titration as a control. These two protocols have to be done in parallel.
- + Reasonable duration of the experiment.
- + This protocol can be extended with the Complex IV module in the following protocols: SUIT-009 O2 mt D015 and SUIT-009 O2 ce-pce D016.
- + Cytochrome c test can be performed in the following protocols: SUIT-009 O2 mt D015 and SUIT-009 O2 ce-pce D016, but not simultaneously with the fluorescence assay.
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, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
|MiPNet24.10 H2O2 flux analysis||2020-05-18|
|MiPNet20.14 AmplexRed H2O2-production||2019-06-24||Mouse||Heart|
|Komlodi 2018 Methods Mol Biol||2018||Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55.||Human|
|Komlodi 2018 J Bioenerg Biomembr||2018||Komlódi T, Geibl FF, Sassani M, Ambrus A, Tretter L (2018) Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria. J Bioenerg Biomembr 10.1007/s10863-018-9766-8.||Guinea pig||Heart|
|Makrecka-Kuka 2015 Biomolecules||2015||Human|
|Krumschnabel 2015 Methods Mol Biol||2015||Mouse||Nervous system|
MitoPedia methods: Fluorometry