SUIT-010 O2 ce-pce D008

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high-resolution terminology - matching measurements at high-resolution

SUIT-010 O2 ce-pce D008


Respirometric test of optimum digitonin concentration

Abbreviation: Dig titration-pce

Reference: A -SUIT-010 - Optimum digitonin concentration for pce

SUIT number: D008_ce1;ce2Rot;ce3S;ce4D;1Dig;1c

O2k-Application: O2

SUIT-010 O2 ce-pce D008 is designed for the evaluation of optimum digitonin concentration for selective cell membrane permeabilization, a requirement to account for differences between cell types, the concentration of cells, and variability between batches of 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 within optimal digitonin concentration and inhibited by over-titration with digitonin, since the mitochondrial outer membrane is affected and cytochrome c can be released. Therefore, it is recommended to titrate cytochrome c after the optimal digitonin concentration has 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, Huete-Ortega M and Gnaiger E (last update 2019-06-05)

Representative traces


MitoPedia: SUIT

Steps and respiratory states


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


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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

Chemicals and syringes

Step Chemical(s) and link(s) Comments
ce2Rot Rotenone (Rot)
ce3S Succinate (S)
ce4D ADP (D)
1Dig Digitonin (Dig)
1c Cytochrome c (c)
Suggested stock concentrations are shown in the specific DL-Protocol.


Doerrier 2018 Methods Mol Biol2018Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, MΓ©szΓ‘ros 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.
Saccharomyces cerevisiae
Skeletal muscle
Endothelial;epithelial;mesothelial cell
Blood cells

MitoPedia concepts: SUIT protocol, SUIT A, Find 

MitoPedia methods: Respirometry 



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