SUIT-018 AmR mt D031

high-resolution terminology - matching measurements at high-resolution

SUIT-018 AmR mt D031

Description

Abbreviation: O₂ dependence-H₂O₂

Reference: A -SUIT-018 - SUIT protocol for simultaneous determination of O₂ and H₂O₂ flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) at low oxygen concentration (tissue normoxia)

SUIT number: D031_mt;1GMS;2D;3Ama

O2k-Application: AmR

SUIT-018 AmR mt D031 is a short protocol to study simultaneously the oxygen dependence of O₂ flux and H₂O₂ production on mitochondrial preparations. Is it possible to run the protocol under different oxygen concentration conditions: 1.) under tissue normoxia (50-60 µM = [O₂]), 2.) under tissue hyperoxia (> 120 µM = [O₂]) or 3.) you can change the oxygen concentration during the experiment. To decrease or increase the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration. We recommend to run SUIT-018 AmR mt D040 simultaneously at high oxygen concentration as a control.

Communicated by  Komlodi T, Gnaiger E (last update 2024-09-19)

Representative traces

Steps and respiratory states

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				AmR or Amplex UltraRed forms resorufin with H₂O₂ catalysed by horseradish peroxidase HRP.

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
1GMS	GMS_L(n)	NS	CI&CII	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	GMS_P	NS	CI&CII	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			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

SUIT-018 AmR mt D040 should be run in parallel (tissue hyperoxia condition).

+ Reasonable duration of the experiment.

+ Simple protocol to evaluate the oxygen dependence of H₂O₂ production in LEAK and OXPHOS.

- Measurements with Amplex UltraRed assay cannot be carried out with liver homogenate.

- Reoxygenation and nitrogen injection could lead to bubble formation in the O2k-Chamber.

- CIV activity and cytochrome c test cannot be performed together with the fluorescence.

Compare SUIT protocols

SUIT-018 AmR mt D041 for simultaneous determination of O₂ and H₂O₂ flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) changing the oxygen concentration in the same protocol.
SUIT-006 AmR mt D048 to investigate the dependence of H₂O₂ flux on the mt-membrane potential on the N-control state in mitochondrial preparations.
SUIT-009 to investigate H₂O₂ production driven by the reverse electron transfer (RET) in isolated mitochondria, tissue homogenate and permeabilized cells.

Chemicals and syringes

Step	Chemical(s) and link(s)	Comments
H₂O₂	Hydrogen Peroxide (H₂O₂)

Step	Chemical(s) and link(s)	Comments
0DTPA	DTPA	This step can be skipped
0SOD	Superoxide Dismutase (SOD)
0HRP	Horseradish peroxidase (HRP)
0AmR	Amplex UltraRed (AmR)

Step	Chemical(s) and link(s)	Comments
1GMS	Glutamate (G), Malate (M) and Succinate (S)
2D	ADP (D)
3Ama	Antimycin A (Ama)

Suggested stock concentrations are shown in the specific DL-Protocol.

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. https://doi.org/10.1007/978-1-4939-2257-4_22	Mouse	Nervous system

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