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Bioblasts - Richard Altmann and MiPArt by Odra Noel
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Digitonin is a mild detergent that permeabilizes plasma membranes selectively due to their high cholesterol content, whereas mt-membranes with lower cholesterol content are affected only at higher concentrations. 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 of cultured cells can be determined directly in a respirometric protocol (see: SUIT-010 O2 ce-pce D008).

Abbreviation: Dig

Reference: Pesta 2012 Methods Mol Biol, MiPNet09.12 O2k-Titrations

Application in HRR

Dig: Digitonin (C56H92O29); Sigma D 5628, 1 g, store at RT; FW = 1229.31
Digitonin from Wako Chemicals

Caution: Toxic!

Preparation of 10 mg/mL (8.1 mM) stock solution (dissolved in DMSO):
  1. Weigh 10 mg of digitonin.
  2. Dissolve in 1 mL DMSO.
  3. Divide into 0.1 mL portions
  4. Store at -20 °C.

O2k Manual Titrations: MiPNet09.12 O2k-Titrations
  • Titration volume: 1 µL using a 10 µL syringe.
  • Final conc. in 2 mL O2k-chamber: 5 µg/mL (4.05 µM; typical optimum concentration for selective plasma membrane permeabilization).
  • Respirometric test of optimum digitonin concentration (Doerrier 2018 Methods Mol Biol)
  1. Incubate cells in the O2k in mt-respiration medium (MiR05 or MiR06) and measure ROUTINE respiration.
  2. Titrate succinate (S, 10 mM) and ADP (D, 2 to 5 mM). Most intact cells are impermeable for S and D.
  3. Titrate rotenone (0.1 µM). CI inhibition in intact cells inhibits the TCA cycle such that succinate is not produced, hence a state close to minimum residual oxygen consumption (ROX) is obtained.
  4. Titrate digitonin using the Titration-Injection microPump (TIP2k) or manual titrations, to observe stepwise stimulation of respiration by increasing availability of S and D to the mitochondria, until a maximum respiration level is reached.

SUITbrowser question: Optimum digitonin concentration

The SUIT protocol SUIT-010 was designed as a test to evaluate the optimum digitonin concentration to permeabilize the cell plasma membrane.
This research question, among others, can be found at the SUITbrowser.

MitoPedia topics: Permeabilization agent