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

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SUIT-018 AmR ce-pce D068

Description

Abbreviation: O₂ dependence-H₂O₂

Reference: A -SUIT-018 - SUIT protocol for simultaneous determination of O₂ and H₂O₂ flux in permeabilized cells at various oxygen concentrations

SUIT number: D068_ce1;1Dig;1GMS;2D;3Ama

O2k-Application: AmR

SUIT-018 AmR ce-pce D068 is a protocol to simultaneously study the oxygen dependence of O₂ flux and H₂O₂ flux in permeabilized cells, and to test the chemical inhibitory effect of the AmR assay (SOD, HRP and AmR) on ROUTINE-respiration of living cells. 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 are added in ROUTINE. However, 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. To decrease or increase the oxygen concentration in the O2k-Chamber, see Setting the oxygen concentration.

Communicated by  Komlodi T, Cecatto C and Gnaiger E (last update 2020-06-09)

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 flux in LEAK and OXPHOS.

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

- Reoxygenation and nitrogen injection can 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 such as isolated mitochondria, tissue homogenate and permeabilized cells at different oxygen concentrations in the same protocol.

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
1Dig	Digitonin (Dig)

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

MitoPedia concepts: SUIT protocol, SUIT A, Find

MitoPedia methods: Fluorometry

@@ Line 1: / Line 1: @@
 {{MitoPedia
 |abbr=O<sub>2</sub> dependence-H<sub>2</sub>O<sub>2</sub>
-|description=[[File:Ce1;1Dig;1GMS;2D;3Ama.png|290px]]
+|description=[[File:Ce1;1Dig;2GMS;3D;4Ama.png|290px]]
-|info='''A''' -'''[[SUIT-018]]'''
+|info='''A''' -'''[[SUIT-018]]''' - 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 concentrations
 |application=AmR
 |SUIT number=D068_ce1;1Dig;1GMS;2D;3Ama
 }}
-::: '''[[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 simultaneously study the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> flux in permeabilized cells, and to test the chemical inhibitory effect of the AmR assay ([[SOD]], [[HRP]] and [[Amplex UltraRed | AmR]]) on ROUTINE-respiration of living cells. 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]] are added in ROUTINE. However, 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. To decrease or increase the oxygen concentration in the O2k-Chamber, see [[Setting the oxygen concentration]].
-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 and to test the chemical inhibitory effect of the AmR assay ([[SOD]], [[HRP]] and [[Amplex UltraRed | AmR]] 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__
-  Communicated by  [[Komlodi T]] and [[Gnaiger E]] (last update 2020-02-26)
+  Communicated by  [[Komlodi T]], [[Cecatto C]] and [[Gnaiger E]] (last update 2020-06-09)
 == Representative traces ==
@@ Line 22: / Line 21: @@
 == Strengths and limitations ==
-:::+ 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]].
+:::+ Simple protocol to evaluate the oxygen dependence of H<sub>2</sub>O<sub>2</sub> flux 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 [[ROUTINE-respiration | ROUTINE]] respiration.
-:::- Reoxygenation and nitrogen injection could lead to bubbles formation in the O2k-Chamber.
+:::- Reoxygenation and nitrogen injection can 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| mitochondrial preparations]] such as isolated mitochondria, tissue homogenate and permeabilized cells at different oxygen concentration ranges 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 concentrations 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.
+== Chemicals and syringes ==
+{{Template:Chemicals AmR}}
+{{Template:Chemicals ce}}
+{{Template:Chemicals SUIT-018}}
+::: Suggested stock concentrations are shown in the specific DL-Protocol.
 == References ==