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Difference between revisions of "Digitonin"

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{{MitoPedia
{{MitoPedia
|abbr=Dig
|abbr=Dig
|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.
|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 (see: [[SUIT-010 O2 ce-pce D008]]).
|info=[[Pesta 2012 Methods Mol Biol]], [[MiPNet09.12 O2k-Titrations]]
|info=[[Pesta 2012 Methods Mol Biol]], [[MiPNet09.12 O2k-Titrations]]
}}
}}
{{MitoPedia topics
__TOC__
|mitopedia topic=Permeabilization agent
 
}}
== Application in [[HRR]] ==
== Application in [[HRFR]] ==
 
::: '''Dig: Digitonin''' (C<sub>56</sub>H<sub>92</sub>O<sub>29</sub>); Sigma D 5628, 1 g, store at RT; FW = 1229.31
{{Chemical_description
|abbr=Dig
|trivial name=Digitonin
|complete name=
|chem formula=C<sub>56</sub>H<sub>92</sub>O<sub>29</sub>
|molar mass=1229.31
|vendor=Sigma-Aldrich
|product number=D5628
|store at=4°C
|sensitivity=
|cas=11024-24-1
|h statements=H301, H373
|h info=
toxic if swallowed, may cause damage to organs through prolonged or repeated exposure
}}<!--::: '''Dig: Digitonin''' (C<sub>56</sub>H<sub>92</sub>O<sub>29</sub>); Sigma D 5628, 1 g, store at RT; M = 1229.31 g·mol<sup>-1</sup>-->
:::: [https://www.fishersci.com/shop/products/digitonin-1g/nc0141730 Digitonin from Wako Chemicals]
<!--:::: <span style="color:#8B008B"> '''Caution:''' Toxic!</span>-->
 
 
::::'''Preparation of 10 mg/mL (8.1 mM) stock solution (dissolved in DMSO)''' for '''2-mL O2k-chamber''':
::::# Weigh 10 mg of digitonin.
::::# Dissolve in 1 mL DMSO.
::::# Divide into 0.1 mL portions
::::# Store at -20 °C.


::: [https://www.fishersci.com/shop/products/digitonin-1g/nc0141730 Digitonin from Wako Chemicals]
:::» '''O2k Manual Titrations:'''  [[MiPNet09.12 O2k-Titrations]]


::::* Titration volume ('''2-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
::::* Final concentration: 5 µg/mL (4.05 µM; typical optimum concentration for selective plasma membrane permeabilization).


<span style="color:#8B008B"> '''Caution:''' Toxic!</span>




:::'''Preparation of 8.1 mM stock solution (dissolved in DMSO)''':
::::'''Preparation of 2.5 mg/mL (2.025 mM) stock solution (dissolved in DMSO)''' for '''0.5-mL O2k-chamber''':
::::# Weigh 10 mg of digitonin.
::::# Weigh 2.5 mg of digitonin.
::::# Dissolve in 1 ml DMSO.
::::# Dissolve in 1 mL DMSO.
::::# Divide into 0.1 ml portions
::::# Divide into 0.1 mL portions
::::# Store at -20 °C.
::::# Store at -20 °C.


:::» '''O2k Manual Titrations:'''  [[MiPNet09.12 O2k-Titrations]]


::: '''O2k Manual Titrations:''' [[MiPNet09.12 O2k-Titrations]]
::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL using a 10 µL Hamilton syringe.
::::* Final concentration: 5 µg/mL (4.05 µM; typical optimum concentration for selective plasma membrane permeabilization).


::::* 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 ([[Doerrier 2018 Methods Mol Biol]]) ==
::::* '''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.
::::# 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), succinate (S, 10 mM) and ADP (D, 2 to 5 mM). C<sub>I</sub> inhibition by rotenone inhibits the TCA cycle such that succinate is not produced and most intact cells are impermeable for S and D. 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 increasing availability of S and D to the mitochondria, until a maximum respiration level is reached.
::::# 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.
::::# Titrate cytochrome ''c'' (10 µM) to test for integrity of the mitochondrial outer membrane.
::::* Examples: [[Talk:Digitonin#Examples_for_optimization_of_digitonin_for_permeabilizing_cells| Permeabilizing cells]]
 
 
 
== [[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 [https://suitbrowser.oroboros.at/ SUITbrowser].
 


::::* Examples: [[Talk:Digitonin#Examples_for_optimization_of_digitonin_for_permeabilizing_cells| Permeabilizing cells]]
{{MitoPedia topics
|mitopedia topic=Permeabilization agent
}}

Latest revision as of 13:10, 5 May 2022


high-resolution terminology - matching measurements at high-resolution


Digitonin

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 (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-Aldrich: D5628, store at 4°C, CAS: 11024-24-1, M = 1229.31 g·mol-1
Hazard statements: H301, H373; toxic if swallowed, may cause damage to organs through prolonged or repeated exposure
Digitonin from Wako Chemicals


Preparation of 10 mg/mL (8.1 mM) stock solution (dissolved in DMSO) for 2-mL O2k-chamber:
  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 (2-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
  • Final concentration: 5 µg/mL (4.05 µM; typical optimum concentration for selective plasma membrane permeabilization).


Preparation of 2.5 mg/mL (2.025 mM) stock solution (dissolved in DMSO) for 0.5-mL O2k-chamber:
  1. Weigh 2.5 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 (0.5-mL O2k-chamber): 1 µL using a 10 µL Hamilton syringe.
  • Final concentration: 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 rotenone (0.1 µM), succinate (S, 10 mM) and ADP (D, 2 to 5 mM). CI inhibition by rotenone inhibits the TCA cycle such that succinate is not produced and most intact cells are impermeable for S and D. Hence a state close to minimum residual oxygen consumption (ROX) is obtained.
  3. 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.
  4. Titrate cytochrome c (10 µM) to test for integrity of the mitochondrial outer membrane.


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