SUIT-018 AmR ce-pce D068

high-resolution terminology - matching measurements at high-resolution

SUIT-018 AmR ce-pce D068

Description

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 concentration.

SUIT protocol pattern: ce1;1Dig;1GMS;2D-

SUIT-018 AmR ce-pce D068 is a protocol to study simultaneously the oxygen dependence of O₂ flux and H₂O₂ production on permeabilized cells at various oxygen concentrations. The oxygen concentration in the chamber can be altered injecting nitrogen with a syringe. To test the inhibitory effect of the AmR assay, DTPA, SOD, HRP and AmR were added in ROUTINE. The inhibitory effect of the AmR assay can be tested in separate protocols: SUIT-003 AmR ce D058 and SUIT-003 AmR ce D059.

Communicated by  Komlodi T and Gnaiger E (last update 2020-02-26)

Representative traces

Steps and respiratory states

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

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 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-009 AmR ce-pce D019 to investigate H₂O₂ production driven by the reverse electron transfer (RET) in permeabilized cells.

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

MitoPedia concepts: SUIT protocol, SUIT A, Find 

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