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Difference between revisions of "SUIT-003 AmR ce D059"

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{{MitoPedia
{{MitoPedia
|abbr=ce
|abbr=AmR effect on ce - control
|description=[[File:1ce;2ceOmy;3ceU-.jpg|150px]]
|description=[[File:ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama.png|500px]]
|info='''B:''' Coupling control in intact cells with oligomycin to study the inhibitory effect of the AmR assay components on the respiration ; ce: non-permeabilized cells -'''[[SUIT-003]]'''
|info='''B:''' Experimental control for [[SUIT-003 AmR ce D058]]  
|application=AmR
|application=AmR
|SUIT number=D059_ce1;ce2SOD;ce3HRP;ce4DMSO;ce5Omy;ce6U;ce7Rot;ce8Ama
|SUIT number=D059_ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama
}}
}}
::: '''[[MitoPedia: SUIT]]'''
::: '''[[MitoPedia: SUIT]]''' - Carrier control protocol to [[SUIT-003 AmR ce D058]]
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' ce
::: '''[[SUIT protocol pattern]]:''' ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama
::: '''[[SUIT protocol pattern]]:''' linear cell (ce) [[coupling control protocol]] ce1;ce2Omy;ce3U
 
 
SUIT-003 AmR ce D059 is a control protocol of [[SUIT-003 AmR ce D058]] to study the effect of the [[Carrier control titrations|carrier titrations]] on the respiration in a parallel experiment.
SUIT-003 AmR ce D059 and SUIT-003 AmR ce D058 were designed to study the inhibitory effect of the components of the Amplex UltraRed assay on [[coupling control state]] of living cells. The different capacities are tested in the sequence [[OXPHOS]], [[LEAK]] and [[ET]]. Also, in order to study the [[LEAK-respiration]], we need to inhibit the [[phosphorylation system]] which in our protocol is achieved using [[oligomycin]]. However, the final concentration of [[oligomycin]] has to be carefully adjusted in preliminary experiments for different samples and, the inhibitory effect on the [[Electron transfer-pathway|electron transfer system]], has to be studied to avoid an underestimation of the [[ET-capacity]]. [[Superoxide dismutase]] and [[Horseradish peroxidase]] do not inhibit respiration, however, [[Amplex UltraRed]] seems to block at higher concentration [[ROUTINE]] respiration and the [[ET-capacity]]. The inhibitory effect of [[Amplex UltraRed]] should be tested with each sample type. SUIT-003 AmR ce D058 shows the titration of the AmR assay, while SUIT-003 AmR ce D059 serves as a carrier control titration; these protocols should be run simultaneously.


{{Template:SUIT text D059}}


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  Communicated by  [[Garcia-Souza LF]], [[Komlodi T]] and [[Gnaiger E]](last update 2019-06-26)
  Communicated by  [[Garcia-Souza LF]] (last update 2019-05-20)




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== Strengths and limitations ==
== Strengths and limitations ==
:::* This protocol has been designed to study the inhibitory effect of the Amplex UltraRed assay.  
::* This protocol has been designed to study the effect of the [[Carrier control titrations|carrier titrations]] on the respiration in parallel to [[SUIT-003 AmR ce D058]]. Therefore, instead of SOD, HRP and AmR, the carriers H<sub>2</sub>O; MiR05; and DMSO are titrated.
:::* It is recommended to run this test with every different sample type.
::* It is recommended to run this test with every different sample type used.
:::+ Reasonable duration of the experiment.
::+ Reasonable duration of the experiment.
:::- The concentration of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[ET-capacity]].
::- The concentration of [[oligomycin]] has to be carefully tested to avoid inhibitory effects in the evaluation of the [[ET capacity]].
:::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.
::- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.


== Compare SUIT protocols ==
== Compare SUIT protocols ==


::::* [[SUIT-003 AmR ce D058]]: is a protocol to study the inhibitroy effect of the AmR assay towards the respiration  
::* [[SUIT-003 AmR ce D058]]: is a protocol to study the inhibitory effect of the AmR assay towards the respiration  
::::* [[SUIT-003 O2 ce D009]] for non-permeabilized cells with oligomycin without the AmR assay
::* [[SUIT-003 O2 ce D009]] for non-permeabilized cells with oligomycin without the AmR assay
::::* Protocols using AmR assay:
:::::* [[SUIT-006 AmR mt D048]] to study simultaneously the O<sub>2</sub> flux and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells, in particular to study the influence of mt-membrane potential on NADH-linked pathway initiated mt-H2O2 production.
:::::*[[SUIT-009 AmR ce-pce D019]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux for non-permeabilized (ce) and permeabilized cells (pce; permeabilization of the plasma membrane occurs in the chambers through addition of digitonin )
:::::* [[SUIT-009 AmR mt D021]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria and tissue homogenate
:::::* [[SUIT-013 AmR ce D023]] for intact cells
:::::*[[SUIT-018 AmR mt D031]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells at low oxygen concentration (tissue normoxia).
:::::* [[SUIT-018 AmR mt D040]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells at high oxygen concentration (tissue hyperoxia).
:::::* [[SUIT-018 AmR mt D041]] simultaneous determination of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> flux on isolated mitochondria, tissue homogenate and permeabilized cells changing the oxygen concentration in the same protocol.


== References ==
== References ==
{{#ask:[[Category:Publications]] [[Additional label::SUIT-003 AmR ce D058]] [[Instrument and method::O2k-Protocol]]
{{#ask:[[Category:Publications]] [[Additional label::SUIT-003 AmR ce D059]] [[Instrument and method::O2k-Protocol]]
|?Was published in year=Year
|?Was published in year=Year
|?Has title=Reference
|?Has title=Reference

Latest revision as of 13:44, 7 July 2020


high-resolution terminology - matching measurements at high-resolution


SUIT-003 AmR ce D059

Description

Ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama.png

Abbreviation: AmR effect on ce - control

Reference: B: Experimental control for SUIT-003 AmR ce D058

SUIT number: D059_ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama

O2k-Application: AmR

MitoPedia: SUIT - Carrier control protocol to SUIT-003 AmR ce D058
SUIT protocol pattern: ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama

SUIT-003 AmR ce D059 is a control protocol of SUIT-003 AmR ce D058 to study the effect of the carrier titrations on the respiration in a parallel experiment. This protocol should be run in parallel with SUIT-003 AmR ce D058 to compare the respiratory capacities and the Flux Control Ratios (FCR) in order to detect any inhibition of the respiratory capacities by the Amplex UltraRed or other components of the ROS assay.

Communicated by  Garcia-Souza LF, Komlodi T and Gnaiger E(last update 2019-06-26)


MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama.png

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.
ce1H2O ROUTINE ce1;ce1H2O
  • SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
ce1MiR05 ROUTINE ce1;ce1H2O;ce1MiR05
  • HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
ce1DMSO ROUTINE ce1;ce1H2O;ce1MiR05;ce1DMSO
ce2Omy LOmy ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U E ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U
ce4Rot ROX ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.
ce5Ama ROX ce1;ce1H2O;ce1MiR05;ce1DMSO;ce2Omy;ce3U;ce4Rot;ce5Ama
  • 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

  • This protocol has been designed to study the effect of the carrier titrations on the respiration in parallel to SUIT-003 AmR ce D058. Therefore, instead of SOD, HRP and AmR, the carriers H2O; MiR05; and DMSO are titrated.
  • It is recommended to run this test with every different sample type used.
+ Reasonable duration of the experiment.
- The concentration of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET capacity.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

References

MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry