(Created page with "== Application in HRR == ::: '''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 ::: [https://www.fishersci.co...") ย |
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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
- Caution: Toxic!
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.
- Dissolve in 1 mL DMSO.
- Divide into 0.1 mL portions
- Store at -20 ยฐC.
- Preparation of 10 mg/mL (8.1 mM) stock solution (dissolved in DMSO) for 2-mL O2k-chamber:
- 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:
- Weigh 2.5 mg of digitonin.
- Dissolve in 1 mL DMSO.
- Divide into 0.1 mL portions
- Store at -20 ยฐC.
- Preparation of 2.5 mg/mL (2.025 mM) stock solution (dissolved in DMSO) for 0.5-mL O2k-chamber:
- 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)
- 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 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.
- 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.
- Examples: Permeabilizing cells