Difference between revisions of "Digitonin"
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::# Titrate succinate (S, 10 mM) and ADP (D, 2 to 5 mM). Most intact cells are impermeable for S and D. | ::# Titrate succinate (S, 10 mM) and ADP (D, 2 to 5 mM). Most intact cells are impermeable for S and D. | ||
::# Titrate rotenone (0.1 µM). C<sub>I</sub> 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. | ::# Titrate rotenone (0.1 µM). C<sub>I</sub> 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. | ||
::# Titrate digitonin using the Titration-Injection microPump (TIP2k) or manual titrations, to observe stepwise stimulation of respiration by | ::# 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 plateaux of maximum respiraiton is reached. | ||
Examples are show here: [[Talk:Digitonin#Examples_for_optimization_of_digitonin_for_permeabilizing_cells| Permeabilizing cells]] |
Revision as of 13:02, 13 November 2015
Description
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.
Abbreviation: Dig
Reference: Pesta 2012 Methods Mol Biol, MiPNet09.12 O2k-Titrations
MitoPedia methods:
Respirometry
MitoPedia topics:
Permeabilization agent
Application in HRR
Dig: Digitonin (C56H92O29); Sigma D 5628, 1 g, store at RT; FW = 1229.31
Caution: Toxic!
Preparation of 8.1 mM stock solution (dissolved in DMSO):
- 1. Weigh 10 mg of digitonin.
- 2) Dissolve in 1 ml DMSO.
- 3) 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: 4.1 µM (typical optimum concentration for selective plasma membrane permeabilization).
- Respirometric test of optimum digitonin concentration (Pesta 2012 Methods Mol Biol)
- Incubate cells in the O2k in mt-respiration medium (MiR05 or MiR06) and measure ROUTINE respiration.
- Titrate succinate (S, 10 mM) and ADP (D, 2 to 5 mM). Most intact cells are impermeable for S and D.
- 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.
- 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 plateaux of maximum respiraiton is reached.
Examples are show here: Permeabilizing cells