SUIT-009 AmR mt D021

high-resolution terminology - matching measurements at high-resolution

SUIT-009 AmR mt D021

Description

Abbreviation: NS(P)

Reference: A: SUIT-009 short protocol for H₂O₂ (AmR) in mitochondrial preparations (isolated mitochondria and tissue homogenate).

SUIT number: D021_1S;2D;3P;4Rot;5Ama

O2k-Application: AmR

SUIT-category: NS(P)

SUIT protocol pattern: orthogonal 1S;2D;3P;4Rot

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

SUIT protocols

• SUIT-009 AmR mt D021
• SUIT-009 AmR pce D019
• SUIT-009 O2 mt D015
• SUIT-009 O2 pce D016

Description

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.

For O2-Application, choose SUIT-009 O2 mt D015; for O2 and H₂O₂ measurements (AmR), choose: D021_1S;2D;3P;4Rot;5Ama.

References

	Year	Reference	Organism	Tissue;cell
MiPNet24.10 H2O2 flux analysis	2021-10-22	Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
Komlodi 2021 BEC AmR-O2	2021	Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi.org/10.26124/bec:2021-0004	Saccharomyces cerevisiae	Other cell lines
MiPNet20.14 AmplexRed H2O2-production	2019-06-24	O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed.	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 Mouse	Skeletal muscle HEK
Makrecka-Kuka 2015 Biomolecules	2015	Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. https://doi.org/10.3390/biom5031319	Human Mouse	Heart Nervous system HEK
Krumschnabel 2015 Methods Mol Biol	2015	Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61.	Mouse	Nervous system

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.

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Fluorometry