User:Spitzer Gudrun/Digitonin: Difference between revisions

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


=== 2-mL O2k-chamber: ===
==== 2-mL O2k-chamber: ====
::::'''Preparation of 10 mg/mL (8.1 mM) stock solution (dissolved in DMSO)''' for '''2-mL O2k-chamber''':
::::'''Preparation of 10 mg/mL (8.1 mM) stock solution (dissolved in DMSO)''' for '''2-mL O2k-chamber''':
::::# Weigh 10 mg of digitonin.
::::# Weigh 10 mg of digitonin.
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=== 0.5-mL O2k-chamber: ===
==== 0.5-mL O2k-chamber: ====
::::'''Preparation of 2.5 mg/mL (2.025 mM) stock solution (dissolved in DMSO)''' for '''0.5-mL O2k-chamber''':
::::'''Preparation of 2.5 mg/mL (2.025 mM) stock solution (dissolved in DMSO)''' for '''0.5-mL O2k-chamber''':
::::# Weigh 2.5 mg of digitonin.
::::# Weigh 2.5 mg of digitonin.

Revision as of 12:32, 9 November 2020

Application in HRR

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

2-mL O2k-chamber:

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 ยฐC.
ยป O2k Manual Titrations: MiPNet09.12 O2k-Titrations
  • Titration volume (2-mL O2k-chamber): 1 ยตL (10 mg/mL) using a 10 ยตL Hamilton syringe.
  • Final conc. in 2 mL O2k-chamber: 5 ยตg/mL (4.05 ยตM; typical optimum concentration for selective plasma membrane permeabilization).


0.5-mL O2k-chamber:

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 ยฐC.
ยป O2k Manual Titrations: MiPNet09.12 O2k-Titrations
  • Titration volume (0.5-mL O2k-chamber): 1 ยตL (2.5 mg/mL) using a 10 ยตL Hamilton syringe.
  • Final conc. in 2 mL O2k-chamber: 5 ยตg/mL (4.05 ยตM; 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 ยตM). 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.
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