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Difference between revisions of "SUIT-003 AmR ce D058"

From Bioblast
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::: '''[[SUIT protocol pattern]]:''' linear cell (ce) [[coupling control protocol]] ce1;ce2Omy;ce3U
::: '''[[SUIT protocol pattern]]:''' linear cell (ce) [[coupling control protocol]] ce1;ce2Omy;ce3U


SUIT-003 O2 ce D058 has been designed to study the inhibitory effect of the components of the Amplex UltraRed assay on [[coupling control state]] of intact 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]] respiratoin and the [[ET-capacity]]. The inhibitory effect of [[Amplex UltraRed]] should be tested with each sample type.  
SUIT-003 O2 ce D058 has been designed to study the inhibitory effect of the components of the Amplex UltraRed assay on [[coupling control state]] of intact 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]] respiratoin and the [[ET-capacity]]. The inhibitory effect of [[Amplex UltraRed]] should be tested with each sample type. SUIT-003 AmR ce D058a shows the titratoin os the AmR assay,while SUIT-003 AmR ce D058b serves as a carrier control titration; these protocols should be run simultaneously.


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== SUIT-003 AmR ce D058b ==
== SUIT-003 AmR ce D058b ==
[[File:D058 O2 traces control.png|400px]]
[[File:D058 O2 traces control.png|400px]]
::::* '''SUIT-003 AmR ce D058b''': is a control protocol of '''SUIT-003 AmR ce D058a''' to study the effect o the carrier titrations on the respiration in a parallel experiment
::::* '''SUIT-003 AmR ce D058b''': is a control protocol of '''SUIT-003 AmR ce D058a''' to study the effect of the [[Carrier control titrations|carrier titrations]] on the respiration in a parallel experiment
{{Template:SUIT-003 AmR ce D058}}
{{Template:SUIT-003 AmR ce D058}}



Revision as of 10:31, 15 May 2019


high-resolution terminology - matching measurements at high-resolution


SUIT-003 AmR ce D058

Description

Ce1;ce2Omy;ce3U-.png

Abbreviation: ce

Reference: 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

SUIT number: D058_ce1;ce2SOD;ce3HRP;ce4AmR;ce5Omy;ce6U;ce7Rot;ce8Ama

O2k-Application: AmR

MitoPedia: SUIT
SUIT-category: ce
SUIT protocol pattern: linear cell (ce) coupling control protocol ce1;ce2Omy;ce3U

SUIT-003 O2 ce D058 has been designed to study the inhibitory effect of the components of the Amplex UltraRed assay on coupling control state of intact 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 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 respiratoin and the ET-capacity. The inhibitory effect of Amplex UltraRed should be tested with each sample type. SUIT-003 AmR ce D058a shows the titratoin os the AmR assay,while SUIT-003 AmR ce D058b serves as a carrier control titration; these protocols should be run simultaneously.

Communicated by  Komlodi T and Gnaiger E (last update 2019-05-15)

Specific SUIT protocols

SUIT-003 AmR ce D058a

D058 O2 traces AmR.png

  • SUIT-003 AmR ce D058a: is a protocol to study the inhibitroy effect of the AmR assay towards the respiration

SUIT-003 AmR ce D058b

D058 O2 traces control.png

  • SUIT-003 AmR ce D058b: is a control protocol of SUIT-003 AmR ce D058a to study the effect of the carrier titrations on the respiration in a parallel experiment
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1SOD;ce1HRP;ce1AmR;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.
ce1SOD ROUTINE ce1;ce1SOD
  • SOD or superoxide dismutase converts the anion superoxide released by the mitochondria into H2O2, making it accessible to the Amplex UltraRed assay.
ce1HRP ROUTINE ce1;ce1SOD;ce1HRP
  • HRP or horseradish peroxidase catalyses the conversion of Amplex UltraRed and H2O2 towards the fluorescent resorufin.
ce1AmR ROUTINE ce1;ce1SOD;ce1HRP;ce1AmR
ce2Omy LOmy ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
ce3U E ce1;ce1SOD;ce1HRP;ce1AmR;ce2Omy;ce3U
ce4Rot ROX ce1;ce1SOD;ce1HRP;ce1AmR;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;ce1SOD;ce1HRP;ce1AmR;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 inhibitory effect of the Amplex UltraRed assay.
  • It is recommended to run this test with every differetn sample type.
+ 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

  • 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 O2 flux and H2O2 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 O2 and H2O2 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 O2 and H2O2 flux on isolated mitochondria and tissue homogenate
  • SUIT-013 AmR ce D023 for intact cells
  • SUIT-018 AmR mt D031 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells at low oxygen concentration (tissue normoxia).
  • SUIT-018 AmR mt D040 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells at high oxygen concentration (tissue hyperoxia).
  • SUIT-018 AmR mt D041 simultaneous determination of O2 and H2O2 flux on isolated mitochondria, tissue homogenate and permeabilized cells changing the oxygen concentration in the same protocol.

References

 YearReferenceOrganismTissue;cell
MiPNet24.10 H2O2 flux analysis2021-10-22
O2k-Protocols
Hydrogen peroxide flux analysis using Amplex UltraRed assay in MiR05-Kit with DatLab 7.4
Komlodi 2021 BEC AmR-O22021Komlódi 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:2021-0004Saccharomyces cerevisiaeOther cell lines
MiPNet20.14 AmplexRed H2O2-production2019-06-24
O2k-Protocols
O2k-FluoRespirometry: HRR and simultaneous determination of H2O2 production with Amplex UltraRed.
MouseHeart
Komlodi 2018 Methods Mol Biol2018Komlodi 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 Biomolecules2015Makrecka-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/biom5031319Human
Mouse
Heart
Nervous system
HEK
Krumschnabel 2015 Methods Mol Biol2015Krumschnabel 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.MouseNervous system


MitoPedia concepts: SUIT protocol, SUIT B, Find 


MitoPedia methods: Respirometry