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Difference between revisions of "SUIT-010"

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{{MitoPedia
{{MitoPedia
|abbr=ceS,S
|abbr=Digitonin test
|description=[[File:ce1;ce2Rot;ce3S;ce4D;1Dig;1c.png|400px|Respirometric test of optimum digitonin concentration ]]
|description=[[File:ce1;ce2Rot;ce3S;ce4D;1Dig;1c.png|400px|Respirometric test of optimum digitonin concentration ]]
|info='''A''' '''Optimization of [[digitonin]] concentration for [[permeabilized cells |pce]]''' [[File:PDF.jpg|100px|link=http://wiki.oroboros.at/images/6/64/SUIT-010.pdf|Bioblast pdf]] »[http://wiki.oroboros.at/index.php/File:SUIT-010.pdf Versions]
|info='''A:''' '''Optimization of [[digitonin]] concentration for [[permeabilized cells |pce]]'''  
}}
}}
::: '''[[Categories of SUIT protocols |SUIT-category]]:''' ceS,S
::: '''[[SUIT protocol pattern]]:''' ce1;ce2Rot;ce3S;ce4D;1Dig;1c
::: '''[[SUIT protocol pattern]]:''' ce1;ce2Rot;ce3S;ce4D;1Dig;1c


SUIT-010 is designed for evaluation of optimum [[digitonin]] concentration for permeabilizing cells, a requirement to account for differendes between cell types, concentration of cells, and variability between batches of the natural product [[digitonin]].
SUIT-010 is designed for the evaluation of optimum [[digitonin]] concentration for permeabilizing cells, a requirement to account for differences between cell types, the concentration of cells, and variability between batches of the natural product [[digitonin]]. After inhibition of the endogenous [[ROUTINE]] respiration by rotenone, respiration of living cells is not stimulated by succinate and ADP. Subsequent stepwise digitonin titration yields gradual permeabilization of the cell membrane, indicating by the increase of respiration up to full permeabilization. Respiration is constant over optimal digitonin concentration and inhibited by over-titration of digitonin, since the mitochondrial outer membrane is affected and [[cytochrome c|cytochrome ''c'']] can be released. Therefore, it is recommended to add [[cytochrome c|cytochrome ''c'']] after the optimal digitonin concentration had been reached. In other SUIT protocols for permeabilized cells, the respiration of pce is stable, if the optimal digitonin concentration is used.  


__TOC__
__TOC__
  Communicated by [[Cardoso LH]], [[Iglesias-Gonzalez J]], [[Huete-Ortega M]] and [[Gnaiger E]] (last update 2019-01-29)
  Communicated by [[Doerrier C]], [[Komlodi T]], [[Cardoso LHD]], [[Huete-Ortega M]], [[Gnaiger E]] (last update 2019-06-05)
 
== Specific SUIT protocols ==
== Specific SUIT protocols ==
=== SUIT-010 O2 ce-pce D008 ===
[[File:ce1;ce2Rot;ce3S;ce4D;1Dig;1c.png|400px|Respirometric test of optimum digitonin concentration ]] [[File:SUIT-010.png|500px]]
[[File:ce1;ce2Rot;ce3S;ce4D;1Dig;1c.png|400px|Respirometric test of optimum digitonin concentration ]] [[File:SUIT-010.png|500px]]
* [[SUIT-010 O2 pce D008]]
* [[SUIT-010 O2 ce-pce D008]] is a protocol to test the optimum concentration of [[digitonin]]
 
 
{{Template:SUIT-010}}
 
== Strengths and limitations ==
:::* [[Digitonin]] is a natural product and thus the effective concentration has to be determined by titrations for every batch. The optimum effective digitonin concentrations for complete plasma membrane permeabilization can be determined with this protocol.
:::* Plasma membrane permeabilization is faster than mitochondrial isolation.
:::* This protocol might be used simultaneously for selecting optimum experimental cell concentrations.


== Compare SUIT protocols ==
::::* [[SUIT-003]] have been designed to study the coupling control state of living cells
::::* [[SUIT-003 O2 ce D028]]  [[coupling-control protocol]] with succinate for permeability test
::::* [[SUIT-006]] protocol is a [[coupling-control protocol]] for [[mitochondrial preparations]]


== References ==
== References ==
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|order=descending
|order=descending
}}
}}
{{Template:SUIT-010}}
== Strengths and limitations ==
:::* [[Digitonin]] is a natural product and thus the effective concentration has to be determined by titrations for every batch. The optimum effective digitonin concentrations for complete plasma membrane permeabilization can be determined with this protocol.
== Compare SUIT protocols ==
::::* [[SUIT-003 O2 ce D028]]
::::* [[SUIT-003]]
::::* [[SUIT-006]]
{{MitoPedia concepts
{{MitoPedia concepts
|mitopedia concept=MiP concept, SUIT protocol, Recommended
|mitopedia concept=MiP concept, SUIT protocol, Recommended

Latest revision as of 22:54, 12 April 2021


high-resolution terminology - matching measurements at high-resolution


SUIT-010

Description

Respirometric test of optimum digitonin concentration

Abbreviation: Digitonin test

Reference: A: Optimization of digitonin concentration for pce

SUIT protocol pattern: ce1;ce2Rot;ce3S;ce4D;1Dig;1c

SUIT-010 is designed for the evaluation of optimum digitonin concentration for permeabilizing cells, a requirement to account for differences between cell types, the concentration of cells, and variability between batches of the natural product digitonin. After inhibition of the endogenous ROUTINE respiration by rotenone, respiration of living cells is not stimulated by succinate and ADP. Subsequent stepwise digitonin titration yields gradual permeabilization of the cell membrane, indicating by the increase of respiration up to full permeabilization. Respiration is constant over optimal digitonin concentration and inhibited by over-titration of digitonin, since the mitochondrial outer membrane is affected and cytochrome c can be released. Therefore, it is recommended to add cytochrome c after the optimal digitonin concentration had been reached. In other SUIT protocols for permeabilized cells, the respiration of pce is stable, if the optimal digitonin concentration is used.

Communicated by Doerrier C, Komlodi T, Cardoso LHD, Huete-Ortega M, Gnaiger E (last update 2019-06-05)

Specific SUIT protocols

SUIT-010 O2 ce-pce D008

Respirometric test of optimum digitonin concentration SUIT-010.png


MitoPedia: SUIT

Steps and respiratory states

SUIT-010

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.
ce2Rot ROX ce1;ce2Rot
ce3S SL(n) S CII ce1;ce2Rot;ce3S
ce4D SP S CII ce1;ce2Rot;ce3S;ce4D
1Dig SP S CII ce1;ce2Rot;ce3S;ce4D;1Dig
1c SP S CII ce1;ce2Rot;ce3S;ce4D;1Dig;1c


Questions.jpg


Click to expand or collaps

Strengths and limitations

  • Digitonin is a natural product and thus the effective concentration has to be determined by titrations for every batch. The optimum effective digitonin concentrations for complete plasma membrane permeabilization can be determined with this protocol.
  • Plasma membrane permeabilization is faster than mitochondrial isolation.
  • This protocol might be used simultaneously for selecting optimum experimental cell concentrations.

Compare SUIT protocols

References

 YearReferenceOrganismTissue;cell
Doerrier 2018 Methods Mol Biol2018Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. https://doi.org/10.1007/978-1-4939-7831-1_3Human
Mouse
Rat
Saccharomyces cerevisiae
Heart
Skeletal muscle
Endothelial;epithelial;mesothelial cell
Blood cells
HEK
Platelet

MitoPedia concepts: MiP concept, SUIT protocol, Recommended 


MitoPedia methods: Respirometry