Carbonyl cyanide m-chlorophenyl hydrazone

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Carbonyl cyanide m-chlorophenyl hydrazone


Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U; C9H5ClN4; FW = 204.62) is a protonophore (H+ ionophore) and is used as a potent chemical uncoupler of oxidative phosphorylation. Like all uncouplers, CCCP concentrations must be titrated carefully to evaluated the optimum concentration for maximum stimulation of mitochondrial respiration, particularly to avoid inhibition of respiration at higher CCCP concentrations.

Abbreviation: CCCP

Reference: Lou 2007 Biochem J, Skulachev 1998 Biochim Biophys Acta

Application in HRR

C: CCCP (Carbonyl cyanide 3-chlorophenylhydrazone, C9H5ClN4O); Sigma C 2759, 100 mg, store at -20 °C: FW = 204.62. Recommended due to better handling in room temperature, we use for preparation ethanol in OROBOROS Lab.
Caution: Toxic!
Preparation of 1 mM stock solution (dissolved in Ethanol):
  1. Weigh 1.02 mg of CCCP into a glass vial.
  2. Dissolve in 5 mL ethanol (or DMSO - see note below).
  3. Store in glass vials at -20 °C. Bring to room temperature before opening.
  • Storage options:
  1. Divide into 0.2 mL portions in glass vials.
  2. Store the whole solution in 5 mL glass vial and use subsamples during experiments to avoid contamination of the stock solution.

Solubility: CCCP also can be prepared in DMSO. Soluble in DMSO (5 mg/mL), ethanol (1 mg/mL), and methanol (10 mg/mL). Insoluble in water. DMSO has a freezing point of +18.55 °C (65.4 °F).
CCCP is soluble in ethanol up to 10 mM. Working with CCCP below room temperature is therefore possible. - Heidler J
O2k 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 (or 0.25 µM) µM steps
Caution: Bring to room temperature before filling the 10 µL syringe, which is important to avoid blocking the needle.

NOTE: 0.1 mM stock for mt-preparations with high uncoupler sensitivity; 1 mM stock for mt-preparations with low uncoupler sensitivity, livingcells invarious culture media (e.g.,RPMI, DMEM, EGC) and for TIP2k.

MiPNet News CCCP 2019-05-02

CCCP solution is less yellow coloured than FCCP. This may be important when working with fluorometric applications in parallel with high-resolution respirometry. - Cardoso LHD

MiPNet News CCCP 2014-01-15

CCCP is soluble in ethanol up to 10 mM. Working with CCCP below room temperature is therefore possible. - Heidler J

MiPNet News CCCP 2014-01-10

The temperature of the CCCP stock solution dissolved in DMSO should be increased to room temperature before filling the titration syringe. When the CCCP stock solution is kept on ice, the needle of the Hamilton titration syringe can be easily blocked, causing problems during titrations which may not be detected immediately. When performing experiments below room temperature, the needle of the Hamilton syringe is regularly blocked during injections into the O2k chamber. To avoid such problems, FCCP should be used instead of CCCP at low experimental temperature, or in laboratories at low room temperature that may prevail during expeditions. - Laner V, Gnaiger E

MiPNet News CCCP 2013-07-11

  • We replace FCCP by CCCP in the OROBOROS Mitochondrial Research Laboratory. Up to now, we used and recommended FCCP as an uncoupler in high-resolution respirometry. Recently we compared the uncoupling effect of CCCP and FCCP in our on-going experiments with homogenate preparations of mouse brain and heart. In parallel experiments identical results were obtained on ET capacity and mt-membrane potential. Compared to FCCP, a much higher DNP concentration is required for maximum stimulation of oxygen flux (Steinlechner-Maran 1996 Am J Physiol Cell Physiol). In contrast, the optimum CCCP concentration for maximum oxygen flux was only slightly higher than the optimum FCCP concentration. CCCP is significantly less expensive than FCCP. - Eigentler A, Gnaiger E
  • Bioblast alert 2013(02)


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MitoPedia topics: Uncoupler