Abbreviation: Dig titration-pce
SUIT number: D008_ce1;ce2Rot;ce3S;ce4D;1Dig;1c
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)
Steps and respiratory states
|Step||State||Pathway||Q-junction||Comment - Events (E) and Marks (M)|
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- Pathway control
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.
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Chemicals and syringes
|Step||Chemical(s) and link(s)||Comments|
|1c||Cytochrome c (c)|
- Suggested stock concentrations are shown in the specific DL-Protocol.
|Doerrier 2018 Methods Mol Biol||2018||Doerrier 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. https://doi.org/10.1007/978-1-4939-7831-1_3||Human|
MitoPedia methods: Respirometry