Difference between revisions of "SUIT-018 AmR ce-pce D068"

Revision as of 13:12, 6 March 2020

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 decreased by 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. For further information, see: Amplex UltraRed page.

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	GMS_L(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	GMS_P	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.

+ Inhibitory effect of the AmR assay can be tested in the same protocol in ROUTINE respiration.

- Reoxygenation and nitrogen injection could lead to bubbles 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 such as isolated mitochondria, tissue homogenate and permeabilized cells at different oxygen concentration ranges 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

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Fluorometry

@@ Line 8: / Line 8: @@
 ::: '''[[MitoPedia: SUIT]]''' - SUIT protocol for simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> 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 O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> 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 [[Amplex UltraRed|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]]. For further infomration, see: [[Amplex UltraRed]] page.
+SUIT-018 AmR ce-pce D068 is a  protocol to study simultaneously the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on permeabilized cells at various oxygen concentrations. The oxygen concentration in the chamber can be decreased by injecting nitrogen with a syringe. To test the inhibitory effect of the AmR assay, [[DTPA]], [[SOD]], [[HRP]] and [[Amplex UltraRed|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]]. For further information, ''see'': [[Amplex UltraRed]] page.
 __TOC__
@@ Line 22: / Line 22: @@
 == Strengths and limitations ==
-:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in LEAK and OXPHOS.
+:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> production in [[LEAK-respiration | LEAK]] and [[Oxidative_phosphorylation |OXPHOS]].
+:::+ Inhibitory effect of the AmR assay can be tested in the same protocol in [[ROUTINE-respiration | ROUTINE]]       respiration.
-:::+ Inhibitory effect of the AmR assay can be tested in the same protocol on the ROUTINE respiration.
+:::- Reoxygenation and nitrogen injection could lead to bubbles formation in the O2k-Chamber.
-:::- 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 O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux in mitochondrial preparations (isolated mitochondria, tissue homogenate and permeabilized cells) changing the oxygen concentration in the same protocol.
+:::* [[SUIT-018 AmR mt D041]] for simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux in [[Mitochondrial preparations| mitochondrial preparations]] such as isolated mitochondria, tissue homogenate and permeabilized cells at different oxygen concentration ranges in the same protocol.
 :::* [[SUIT-009 AmR ce-pce D019]] to investigate H<sub>2</sub>O<sub>2</sub> production driven by the [[Reverse electron flow from CII to CI| reverse electron transfer]] (RET) in permeabilized cells.
 == References ==
-{{#ask:[[Category:Publications]] [[Additional label::SUIT-018 AmR mt D031]]  [[Instrument and method::O2k-Protocol]]
+{{#ask:[[Category:Publications]] [[Additional label::SUIT-018 AmR ce-pce D068]]  [[Instrument and method::O2k-Protocol]]
 | ?Was published in year=Year
-| ?Has title=Reference
-| ?Mammal and model=Organism
-| ?Tissue and cell=Tissue;cell
-| format=broadtable
-| limit=5000
-| offset=0
-| sort=Was published in year
-| order=descending
-}}
-{{#ask:[[Category:Abstracts]] [[Additional label::SUIT-020 Fluo mt D033]]
-| ?Was submitted in year=Year
 | ?Has title=Reference
 | ?Mammal and model=Organism