SUIT-010

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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 intact 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 LH, Iglesias-Gonzalez J, 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
  • 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.


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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.Human
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