SUIT-010 O2 ce-pce D050

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SUIT-010 O2 ce-pce D050

Description

Ce1;ce2Rot;ce3S(p);ce3S;ce4D;ce4c;1Dig;2Ama.png

Abbreviation: ceS,S

Reference: A Evaluation of plasma membrane integrity of intact cells -SUIT-010

SUIT number: D050_ce1;ce2Rot;ce3S(p);ce3S;ce4D;ce4c;1Dig;2Ama

O2k-Application: O2

MitoPedia: SUIT
SUIT-category: ceS,S
SUIT protocol pattern: diametral ce1;ce2Rot;ce3S(p);ce3S;ce4D;ce4c;1Dig;2Ama


SUIT-010 O2 ce-pce D050 is designed to the plasma membrane integrity of intact cells upon the stimulation of mitochondria by the plasma membrane-permeable succinate, S(p).


Communicated by Bastos Sant'Anna Silva AC and Gnaiger E (last update 2019-04-05)
MitoPedia: SUIT

Steps and respiratory states

Ce1;ce2Rot;ce3S(p);ce3S;ce4D;ce4c;1Dig;2Ama.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.
ce2Rot ROX ce1;ce2Rot
ce3S(p) S S ce1;ce2Rot;ce3S(p)
ce3S S S CII ce1;ce2Rot;ce3S(p);ce3S
ce4D SP S CII ce1;ce2Rot;ce3S(p);ce3S;ce4D
ce4c SP S CII ce1;ce2Rot;ce3S(p);ce3S;ce4D;ce4c
  • Succinate, S ( type S-pathway to Q).
  • Addition of cytochrome c yields a test for integrity of the mtOM. Stimulation by added cytochrome c would indicate an injury of the mtOM and limitation of respiration in the preceding state without added c due to loss of cytochrome c. Typically, cytochrome c is added immediately after the earliest ADP-activation step (P, OXPHOS-capacity with saturating [ADP]).
  • OXPHOS capacity, P (with saturating [ADP]), active OXPHOS state.
1Dig SP S CII ce1;ce2Rot;ce3S(p);ce3S;ce4D;ce4c;1Dig



Questions.jpg


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Strengths and limitations

  • For the release of succinate in the intracellular space, the prodrug should be cleaved by esterases. This feature is only possible if the plasma membrane is intact.
  • We recommend to perform the experiment in cell culture medium ensuring the integrity of the plasma cell. Nevertheless, after the addition of digitonin and consequent exposure of the mitochondrial membrane to the medium, normalization by digitonin step can be questionable.
+ The protocol can be used to compare the utilization of succinate in models of Complex I deficiency.

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.Human
Mouse
Rat
Saccharomyces cerevisiae
Heart
Skeletal muscle
Endothelial;epithelial;mesothelial cell
Blood cells
HEK
Platelet
Ehinger 2016 Nat Commun2016Ehinger JK, Piel S, Ford R, Karlsson M, Sjövall F, Frostner EÅ, Morota S, Taylor RW, Turnbull DM, Cornell C, Moss SJ, Metzsch C, Hansson MJ, Fliri H, Elmér E (2016) Cell-permeable succinate prodrugs bypass mitochondrial complex I deficiency. Nat Commun 7:12317.Heart
Blood cells
Fibroblast

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry 


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SUIT-010