Difference between revisions of "Carbonyl cyanide m-chlorophenyl hydrazone"

Latest revision as of 18:22, 12 September 2022

high-resolution terminology - matching measurements at high-resolution

Carbonyl cyanide m-chlorophenyl hydrazone

Description

Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U; C₉H₅ClN₄; F_W = 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

CCCP: m-Cl-CCP (Carbonyl cyanide 3-chlorophenylhydrazone; C₉H₅ClN₄O), Sigma-Aldrich: C2759, store at -20 °C, CAS: 555-60-2, M = 204.62 g·mol^-1

Hazard statements: H301, H311, H331, H315, H319, H335; toxic if swallowed, toxic in contact with skin, toxic if inhaled, causes skin irritation, causes serious eye irritation, may cause respiratory irritation

Solubility: CCCP also can be prepared in DMSO as well as EtOH. Soluble in DMSO (5 mg/mL), ethanol (1 mg/mL), and methanol (10 mg/mL). Insoluble in water.

Because of better handling at room temperature, we use EtOH in OROBOROS Lab.

Disadvantage of DMSO: freezing point of +18.55 °C (65.4 °F). When using DMSO as solvent, make sure to bring the stock solution to room temperature before filling the 10 µL syringe, which is important to avoid blocking the needle.

Ethanol stock solutions may be stored in 5 mL glass vials (instead of freezing 0.2 mL portions in small vials). Use subsamples during experiments to avoid contamination of the storage solution.

Many labs routinely protect CCCP from light. However, there is no hint on vendor's pages that this is necessary (see Discussion).

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

Preparation of 1 mM stock solution (dissolved in Ethanol) for 2-mL O2k-chamber:

Weigh 1.02 mg of CCCP into a glass vial.
Dissolve in 5 mL ethanol.
Divide into 0.2 mL portions in glass vials and store at -20 °C.

» O2k manual titrations MiPNet09.12 O2k-Titrations

Titration volume (2-mL O2k-chamber): 1 µL (or 0.5 µL) steps using a 10 µL Hamilton syringe.
Final concentration: 0.5 µM (or 0.25 µM) steps

Preparation of 0.25 mM stock solution (dissolved in Ethanol) for 0.5-mL O2k-chamber:

Take 100 µL of 1 mM stock solution.
Dilute with 300 µL ethanol.

» O2k manual titrations MiPNet09.12 O2k-Titrations

Titration volume (0.5-mL O2k-chamber): 1 µL (or 0.5 µL) steps using a 10 µL Hamilton syringe.
Final concentration: 0.5 µM (or 0.25 µM) steps

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)

MitoPedia topics: Uncoupler

Bioblast links: Uncoupling - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>

Specific

» Artefacts by single dose uncoupling

» ATP synthase

» CCCP

» Coupling-control protocol

» DNP

» Dyscoupled respiration

» FCCP

» Is respiration uncoupled - noncoupled - dyscoupled?

» Noncoupled respiration: Discussion

» Uncoupler

» Uncoupled respiration - see » Noncoupled respiration

» Uncoupling proteins

» Uncoupling protein 1

» Uncoupler titrations - Optimum uncoupler concentration

Respiratory states and control ratios

» Biochemical coupling efficiency

» Coupling-control state

» Electron-transfer-pathway state

» Electron-transfer pathway

ET capacity

» E-L coupling efficiency

» Flux control efficiency

» Flux control ratio

» LEAK-control ratio

» LEAK respiration

» Noncoupled respiration

» OXPHOS

» OXPHOS capacity; » State 3

» OXPHOS-control ratio, P/E ratio

» Respiratory acceptor control ratio

» ROUTINE-control ratio

» ROUTINE respiration

» ROUTINE state

» State 3u

» State 4

» Uncoupling-control ratio UCR

Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. Bioenerg Commun 2020.1. https://doi.org/10.26124/bec:2020-0001.v1
Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2. https://doi.org/10.26124/bec:2020-0002

General (alphabetical order)

» Adenine nucleotide translocase

» Adenylates

» Electron transfer pathway

» Mitochondrial preparations

» mt-membrane potential

» Oxygen flux

» Phosphorylation system

» Proton leak

» Proton slip

» TIP2k

Other keyword lists

» Template:Keywords: Force and membrane potential

@@ Line 1: / Line 1: @@
 {{MitoPedia
-|abbr=CCCP, U
+|abbr=CCCP
-|description='''Carbonyl cyanide m-chloro phenyl hydrazone''', CCCP (U;  C<sub>9</sub>H<sub>5</sub>ClN<sub>4</sub>; ''F''<sub>W</sub> = 204.62) is a protonophore (H<sup>+</sup> 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.
+|description='''Carbonyl cyanide m-chlorophenyl hydrazone''', CCCP (U;  C<sub>9</sub>H<sub>5</sub>ClN<sub>4</sub>; ''F''<sub>W</sub> = 204.62) is a protonophore (H<sup>+</sup> 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.
 |info=[[Lou 2007 Biochem J]], [[Skulachev 1998 Biochim Biophys Acta]]
-|type=Uncoupler
-}}
-{{MitoPedia methods
-|mitopedia method=Respirometry
-|type=Uncoupler
-}}
-{{MitoPedia topics
-|mitopedia topic=Uncoupler
-|type=Uncoupler
 }}
+__TOC__
 == Application in [[HRR]] ==
+{{Chemical_description
+|abbr=CCCP
+|trivial name=''m''-Cl-CCP
+|complete name=Carbonyl cyanide 3-chlorophenylhydrazone
+|chem formula=C<sub>9</sub>H<sub>5</sub>ClN<sub>4</sub>O
+|molar mass=204.62
+|vendor=Sigma-Aldrich
+|product number=C2759
+|store at=-20 °C
+|sensitivity=
+|cas=555-60-2
+|h statements=H301, H311, H331, H315, H319, H335
+|h info=toxic if swallowed, toxic in contact with skin, toxic if inhaled, causes skin irritation, causes serious eye irritation, may cause respiratory irritation
+}}<!--::: '''C: CCCP''' (Carbonyl cyanide 3-chlorophenylhydrazone, C<sub>9</sub>H<sub>5</sub>ClN<sub>4</sub>O);  Sigma C 2759, 100 mg, store at -20 °C: M  = 204.62 g·mol<sup>-1</sup>
-=== MiPNet News CCCP 2014-01-15 ===
+:::: <span style="color:#8B008B"> '''Caution:''' Toxic!</span>-->
+::: '''Solubility:''' CCCP also can be prepared in DMSO as well as EtOH. Soluble in DMSO (5 mg/mL), ethanol (1 mg/mL), and methanol (10 mg/mL). Insoluble in water.
+:::: Because of better handling at room temperature, we use EtOH in [[AT_Innsbruck_OROBOROS |OROBOROS Lab]].
+:::: Disadvantage of DMSO: freezing point of +18.55 °C (65.4 °F). When using DMSO as solvent, make sure to bring the stock solution to room temperature before filling the 10 µL syringe, which is important to avoid blocking the needle.
+:::: Ethanol stock solutions may be stored in 5 mL glass vials (instead of freezing 0.2 mL portions in small vials). Use subsamples during experiments to avoid contamination of the storage solution.
+:::: Many labs routinely protect CCCP from light. However, there is no hint on vendor's pages that this is necessary (see Discussion).
+::::'''NOTE''': 0.1 mM stock for mt-preparations with high uncoupler sensitivity; 1 mM stock for mt-preparations with low uncoupler sensitivity, living cells in various culture media (e.g., RPMI, DMEM, EGC) and for TIP2k.
+:::: '''Preparation of 1 mM stock solution''' (dissolved in Ethanol) for '''2-mL O2k-chamber''':
+::::# Weigh 1.02 mg of CCCP into a glass vial.
+::::# Dissolve in 5 mL ethanol<!-- (or DMSO - see note above)-->.
+::::# Divide into 0.2 mL portions in glass vials and store at -20 °C.
-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 ('''2-mL O2k-chamber'''): 1 µL (or 0.5 µL) steps using a 10 µL Hamilton syringe.
+::::* Final concentration: 0.5 µM (or 0.25 µM) steps
-=== 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 [[O2k-Publications: Temperature|low experimental temperature]], or in laboratories at low room temperature that may prevail during [http://www.oroboros.at/?expeditions expeditions].  - [[Laner V]], [[Gnaiger E]]
+:::: '''Preparation of 0.25 mM stock solution''' (dissolved in Ethanol) for '''0.5-mL O2k-chamber''':
+::::# Take 100 µL of 1 mM stock solution.
+::::# Dilute with 300 µL ethanol.
-=== MiPNet News CCCP 2013-07-11 ===
+:::» '''O2k manual titrations'''  [[MiPNet09.12 O2k-Titrations]]
-* '''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 ETS 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]]
+::::* Titration volume ('''0.5-mL O2k-chamber'''): 1 µL (or 0.5 µL) steps using a 10 µL Hamilton syringe.
-* [[Bioblast_alert#Bioblast_alert_2013.2802.29:_2013-08-08|Bioblast alert 2013(02)]]
+::::* Final concentration: 0.5 µM (or 0.25 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 ===
-== Preparation ==
+:::: 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]]
-: '''C: CCCP''' (Carbonyl cyanide 3-chlorophenylhydrazone, C<sub>9</sub>H<sub>5</sub>ClN<sub>4</sub>O);  Sigma C 2759, 100 mg, store at -20 °C: ''F''<sub>W</sub> = 204.62
+=== 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]]
-: '''Preparation of 1 mM stock solution''' (dissolved in DMSO):
+=== MiPNet News CCCP 2014-01-10 ===
-::1) Weigh 1.02 mg of CCCP into a glass vial.
+:::: 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 [[O2k-Publications: Temperature|low experimental temperature]], or in laboratories at low room temperature that may prevail during [[The World as a Laboratory|expeditions]].  - [[Laner V]], [[Gnaiger E]]
-::2) Dissolve in 5 ml DMSO.
-::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.
-::4) Store in glass vials at -20 °C. Bring to room temperature before opening.
-::5) Titrations: Bring to room temperature before filling the 10 µl syringe, which is important to avoid blocking the needle. 1 µl steps, corresponding to 0.5 µM steps of the concentration in the 2 ml O2k-chamber.
+=== 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#Bioblast_alert_2013.2802.29:_2013-08-08|Bioblast alert 2013(02)]]
-<span style="color:#8B008B"> '''Caution:''' Toxic!</span>
+{{MitoPedia concepts}}
+{{MitoPedia methods}}
+{{MitoPedia O2k and high-resolution respirometry}}
+{{MitoPedia topics
+|mitopedia topic=Uncoupler
+}}
+{{Keywords: Uncoupling}}