Difference between revisions of "SUIT-009 AmR ce-pce D019"

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{{MitoPedia
 
{{MitoPedia
|abbr=H<sub>2</sub>O<sub>2</sub> ce-pce
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|abbr=H<sub>2</sub>O<sub>2</sub> RET ce-pce S_L
 
|description=[[File:Ce1;1Dig;1S;2D;3P;4Rot;5Ama.png|520px|SUIT9]]
 
|description=[[File:Ce1;1Dig;1S;2D;3P;4Rot;5Ama.png|520px|SUIT9]]
 
|info='''A''':  short protocol for H<sub>2</sub>O<sub>2</sub> (AmR) in ce: non-permeabilized cells, pce: permeabilized cells  -'''[[SUIT-009]]'''
 
|info='''A''':  short protocol for H<sub>2</sub>O<sub>2</sub> (AmR) in ce: non-permeabilized cells, pce: permeabilized cells  -'''[[SUIT-009]]'''
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|O2k-Application: AmR
 
|O2k-Application: AmR
 
}}
 
}}
::: '''[[MitoPedia: SUIT]]'''
+
::: '''[[SUIT protocol pattern]]: '''1S;2D;3P;4Rot-
::: '''[[SUIT protocol pattern]]:'''1S;2D;3P;4Rot-
+
SUIT-009 is a short protocol for simultaneous determination of O<sub>2</sub> and [[MiPNet20.14 AmplexRed H2O2-production |H<sub>2</sub>O<sub>2</sub>]] flux in [[permeabilized cells]]. Living cells are added first to the O2k chamber and then permeabilized as a part of the protocol. Succinate (S) supports [[reverse electron flow from CII to CI]] (RET) with high H<sub>2</sub>O<sub>2</sub> production in the LEAK state at low O<sub>2</sub> flow, S<sub>''L''</sub>. Respiration is stimulated by ADP (OXPHOS-capacity, S<sub>''P''</sub>), while H<sub>2</sub>O<sub>2</sub> flux declines dramatically. Pyruvate (P) supports the [[NADH Electron transfer-pathway state|NADH-pathway]] and thus the convergent NS-pathway with little further effect on H<sub>2</sub>O<sub>2</sub> flux, NS<sub>''P''</sub>). Malate is not required, since oxaloacetate is formed from succinate-fumarate-malate. Antimycin A (Ama) inhibits Complex III (CIII) at the Q<sub>o</sub> level inhibiting respiration to the level of ''Rox'', and generally increases the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRed® (AmR) changes over experimental time and upon addition of chemicals. To correct H<sub>2</sub>O<sub>2</sub> flux for the sensitivity changes, several [[MiPNet20.14_AmplexRed_H2O2-production#Calibration_with_H2O2|H<sub>2</sub>O<sub>2</sub> calibration steps]] are done during the experiment. The experimental control for O<sub>2</sub> flux in the absence AmR is carried out with [[SUIT-009 O2 ce-pce D016]].
SUIT-009 is a short protocol for simultaneous determination of O<sub>2</sub> flux and the rate of [[MiPNet20.14 AmplexRed H2O2-production|H<sub>2</sub>O<sub>2</sub> production]] (H<sub>2</sub>O<sub>2</sub> flux) in [[Permeabilized cells| permeabilized cells]] (non-permeabilized cells must be added to the chambers and then permeabilized as a part of the protocol). Succinate (S) supports the [[Reverse electron flow from CII to CI| reverse electron transfer]] (RET)-related H<sub>2</sub>O<sub>2</sub> production in the LEAK state. Pyruvate (P) supports [[NADH Electron transfer-pathway state|NADH-pathway (N)]], and usually in the presence of ADP it does not increase further the H<sub>2</sub>O<sub>2</sub> flux. Antimycin A (Ama) inhibits Complex III (CIII) at the Q<sub>o</sub> level and could increase the H<sub>2</sub>O<sub>2</sub> flux. The sensitivity of the Amplex UltraRed® assay (for determining H<sub>2</sub>O<sub>2</sub> production) changes over the experimental time and upon addition of chemicals. To correct H<sub>2</sub>O<sub>2</sub> flux for the sensitivity changes, several [[MiPNet20.14_AmplexRed_H2O2-production#Calibration_with_H2O2|H<sub>2</sub>O<sub>2</sub> calibration steps]] are done during the experiment. The experimental control for the O<sub>2</sub>-Application should be carried out with [[SUIT-009 O2 ce-pce D016]].
 
  
 
__TOC__
 
__TOC__
  Communicated by [[Huete-Ortega M]], [[Cardoso LH]], [[Doerrier C]], [[Iglesias-Gonzalez J]], [[Komlodi T]] and [[Gnaiger E]] (last update 2019-04-01)
+
  Communicated by [[Huete-Ortega M]], [[Cardoso LH]], [[Doerrier C]], [[Iglesias-Gonzalez J]], [[Komlodi T]], [[Gnaiger E]] (last update 2019-06-20)
 
{{Template:SUIT-009 AmR pce D019}}
 
{{Template:SUIT-009 AmR pce D019}}
  
 
== Strengths and limitations ==
 
== Strengths and limitations ==
:::* It is a protocol design to analyse:
+
:::* Information is obtained on respiration and ROS production in the S- and NS-pathway.
:::: 1. the S- and NS-pathway linked respiration and ROS production.
+
:::* [[SUIT-009 O2 ce-pce D016]] determination of O<sub>2</sub> flux on permeabilized cells as a control for SUIT-009 AmR ce-pce D019 to test the inhibitory effect of the fluorescence dye on the mt-respiration.
:::: 2. any Complex I defect, which would lead to a decrease in the N-linked respiration and a reduced reverse electron transfer-supported ROS production.
+
:::* To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: [[SUIT 003 AmR ce D058]] with AmR and [[SUIT 003 AmR ce D059]] with carrier titration as a control. These two protocols should be done in parallel.  
:::: 3. any Complex II defect, which would result in an altered S-linked respiration and ROS production.
+
::: + This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which is inhibited by ADP due to reduction of the protonmotive force, ''pmF''.
:::*[[SUIT-009 O2 ce-pce D016]] determination of O<sub>2</sub> flux on permeabilized cells as a control for SUIT-009 AmR ce-pce D019 to test the inhibitory effect of the fluorescence dye on the mt-respiration.
 
:::* To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: [[SUIT 003 AmR ce D058]] with AmR and [[SUIT 003 AmR ce D059]] with carrier titration as a control. These two protocols have to be done in parallel.  
 
::: + This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which might be inhibited by ADP due to the depolarisation of the mt-membrane.
 
 
::: + Short duration of the experiment.
 
::: + Short duration of the experiment.
  
 
::: - CIV activity and cytochrome ''c'' test cannot be performed together with the fluorescence assay.
 
::: - CIV activity and cytochrome ''c'' test cannot be performed together with the fluorescence assay.
::: - The H<sub>2</sub>O<sub>2</sub> flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of the cytosol.
+
::: - The H<sub>2</sub>O<sub>2</sub> flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of cytosolic compounds.  
::: - In some cell lines e.g. HEK293T, P does not have any effect because of the downregulation of the N-linked pathway.
 
  
  
 
== Compare SUIT protocols ==
 
== Compare SUIT protocols ==
:::* [[SUIT-006 AmR mt D048]] to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on the mt-membrane potantial on the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
+
:::* [[SUIT-006 AmR mt D048]] to investigate the dependence of H<sub>2</sub>O<sub>2</sub> flux on the mt-membrane potential in the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
:::* [[SUIT-018]] is a short protocol to study oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production on isolated mitochondria or tissue homogenate (except of liver).
+
:::* [[SUIT-018]] is a short protocol to study the oxygen dependence of O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> production in isolated mitochondria or tissue homogenate (except of liver).
  
 
== References ==
 
== References ==

Revision as of 06:22, 20 June 2019

Bioblasts - Richard Altmann and MiPArt by Odra Noel
MitoPedia         Terms and abbreviations         Concepts and methods         MitoPedia: SUIT         MiP and biochemistry         Preprints and history



MitoPedia

SUIT-009 AmR ce-pce D019

Description

SUIT9

Abbreviation: H2O2 RET ce-pce S_L

Reference: A: short protocol for H2O2 (AmR) in ce: non-permeabilized cells, pce: permeabilized cells -SUIT-009

SUIT number: D019_ce1;1Dig;1S;2D;3P;4Rot;5Ama

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

SUIT-009 is a short protocol for simultaneous determination of O2 and H2O2 flux in permeabilized cells. Living cells are added first to the O2k chamber and then permeabilized as a part of the protocol. Succinate (S) supports reverse electron flow from CII to CI (RET) with high H2O2 production in the LEAK state at low O2 flow, SL. Respiration is stimulated by ADP (OXPHOS-capacity, SP), while H2O2 flux declines dramatically. Pyruvate (P) supports the NADH-pathway and thus the convergent NS-pathway with little further effect on H2O2 flux, NSP). Malate is not required, since oxaloacetate is formed from succinate-fumarate-malate. Antimycin A (Ama) inhibits Complex III (CIII) at the Qo level inhibiting respiration to the level of Rox, and generally increases the H2O2 flux. The sensitivity of the Amplex UltraRed® (AmR) changes over 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 experimental control for O2 flux in the absence AmR is carried out with SUIT-009 O2 ce-pce D016.

Communicated by Huete-Ortega M, Cardoso LH, Doerrier C, Iglesias-Gonzalez J, Komlodi T, Gnaiger E (last update 2019-06-20)
MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1S;2D;3P;4Rot;5Ama.png

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
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.
1Dig Dig ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1S SL(n) S CII 1S
2D SP S CII 1S;2D
3P NSP NS CI&II 1S;2D;3P
4Rot SP S CII 1S;2D;3P;4Rot
5Ama ROX
  • 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).



Questions.jpg


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Strengths and limitations

  • Information is obtained on respiration and ROS production in the S- and NS-pathway.
  • SUIT-009 O2 ce-pce D016 determination of O2 flux on permeabilized cells as a control for SUIT-009 AmR ce-pce D019 to test the inhibitory effect of the fluorescence dye on the mt-respiration.
  • To study the inhibitory effect of the AmR assay on the respiration using living cells, the following protocols are recommended: SUIT 003 AmR ce D058 with AmR and SUIT 003 AmR ce D059 with carrier titration as a control. These two protocols should be done in parallel.
+ This protocol is designed to evaluate RET in the S-linked pathway in the LEAK state, which is inhibited by ADP due to reduction of the protonmotive force, pmF.
+ Short duration of the experiment.
- CIV activity and cytochrome c test cannot be performed together with the fluorescence assay.
- The H2O2 flux will be much lower compared to that observed with isolated mitochondria, which might be explained by the high antioxidant capacity of cytosolic compounds.


Compare SUIT protocols

  • SUIT-006 AmR mt D048 to investigate the dependence of H2O2 flux on the mt-membrane potential in the N-control state in isolated mitochondria, tissue homogenate and permeabilized cells (already permeabilized when they are added to the chamber).
  • SUIT-018 is a short protocol to study the oxygen dependence of O2 flux and H2O2 production in isolated mitochondria or tissue homogenate (except of liver).

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Fluorometry