Difference between revisions of "FCCP"

Revision as of 06:09, 22 April 2014

high-resolution terminology - matching measurements at high-resolution

FCCP

Description

FCCP (Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone) is a protonophore or uncoupler: FCCP added at uncoupler concentration U_c; c is the optimum uncoupler concentration in titrations to obtain maximum mitochondrial respiration in the noncoupled state of ETS capacity.

Abbreviation: U_c

Reference: MiPNet10.04; Steinlechner-Maran 1996 Am J Physiol Cell Physiol

MitoPedia topics: Uncoupler

CCCP versus FCCP

Bioblast alert 2013(02): We replace FCCP by the less expensive CCCP in our experiments with high-resultion respirometry (HRR).

FCCP should be used instead of CCCP at low experimental temperature, or in laboratories at low room temperature that may prevail during expeditions, to avoid blocking the needle of the Hamilton titration syringe.

Application in HRR

F: FCCP (Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, C₁₀H₅F₃N₄O); Sigma C 2920, 10 mg, store at 4 °C: FW = 254.17)

Caution: Toxic!

Preparation of 1 mM stock solution (dissolved in 99.9% ethanol):

1. Weigh 1.27 mg of FCCP into a glass vial.

2. Dissolve in 5 ml ethanol.

3. Divide into 0.2 ml portions, or store in 5 ml glass vial and use subsamples during experiments to avoid contamination of the storage solution.

4. Store in glass vials at -20 °C.

Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.

Oxygraph-2k manual titrations MiPNet09.12 O2k-Titrations

Titration volume: 1 µl (or 0.5 µl) steps using a 10 µl syringe (2 ml O2k-Chamber).
Final concentration: 0.5 µM (or 0.25 µM) steps.

Frequently, a more diluted FCCP stock is necessary for reducing the concentration steps in manual titrations. In this case prepare more diluted stocksolution (ditution 2-10x) from your storage solution. The highest accuracy of uncoupler titrations is achieved by titrations with the TIP2k at high concentrations of the stock solution. ^[1]

↑ Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley: 327-352. »Bioblast Access

[1] Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley: 327-352. »Bioblast Access

[1]

@@ Line 2: / Line 2: @@
 |abbr=U<sub>''c''</sub>
 |description='''FCCP''' (Carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone) is a protonophore or [[uncoupler]]: '''FCCP''' added at uncoupler concentration U<sub>''c''</sub>; ''c'' is the [[optimum uncoupler concentration]] in titrations to obtain maximum mitochondrial respiration in the [[noncoupled respiration|noncoupled]] state of [[ETS capacity]].
-|info=[[MiPNet10.04]]; [[SteinlechnerMaran_1996_AJP]]
+|info=[[MiPNet10.04]]; [[Steinlechner-Maran 1996 Am J Physiol Cell Physiol]]
 |type=Uncoupler
 }}
@@ Line 23: / Line 23: @@
 <span style="color:#8B008B"> '''Caution:''' Toxic!</span>
-<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.</span>
+'''Preparation of 1 mM stock solution''' (dissolved in 99.9% ethanol):
-'''Preparation of 1 mM stock solution''' (dissolved in 99.9% ethanol):
+::1. Weigh 1.27 mg of FCCP into a glass vial.
+::2. Dissolve in 5 ml ethanol.
+::3. Divide into 0.2 ml portions, or store in 5 ml glass vial and use subsamples during experiments to avoid contamination of the storage solution.
+::4. Store in glass vials at -20 °C.
-::1) Weigh 1.27 mg of FCCP into a glass vial.
+'''Caution:''' Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.
-::2) Dissolve in 5 ml ethanol.
-::3) Divide into 0.2 ml portions OR store in 5 ml glass vial and use subsamples during experiments to avoid contamination of the stock solution.
-::3) Store in glass vials (recommendation) at -20 °C.
+'''Oxygraph-2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
-'''Oxygraph-2k manual titrations:'''  [[MiPNet09.12]]
+::* Titration volume: 1 µl (or 0.5 µl) steps using a 10 µl syringe (2 ml O2k-Chamber).
+::* Final concentration: 0.5 µM (or 0.25 µM) steps.
-::* Titration volume: 1 µl steps using a 10 µl syringe
+Frequently, a more diluted FCCP stock is necessary for reducing the concentration steps in manual titrations. In this case prepare more diluted stocksolution (ditution 2-10x) from your storage solution. The highest accuracy of uncoupler titrations is achieved by titrations with the [[TIP2k]] at high concentrations of the stock solution. <ref> Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley: 327-352. [[Gnaiger_2008_POS |»Bioblast Access]]</ref>
-::* Final conc. in 2 ml O2k-chamber: 0.5 µM steps
-Frequently, more diluted FCCP for getting several titration steps is necessary. In this case prepare more diluted solution (ditution 2-10x) from your stock solution.
+<references/>