3-Cyano-7-ethoxycoumarin is a cytochrome P-450 substrate.

Store at -20°C in dark

Detect P450 Activities in Intact Cells by 3-Cyano-7-ethoxycoumarin:

3-Cyano-7-ethoxycoumarin stock solution preparation:

Dissolve 3-Cyano-7-ethoxycoumarin in DMSO at concentration of 30mM. Store at -20°C in dark.

Incubation medium preparation:

1 mM Na2HPO4

137 mM NaCl

5 mM KCl

0.5 mM MgCl₂

2 mM CaCl₂

10 mM glucose

10 mM Hepes, pH 7.4

Quenching solution preparation:

0.1 M phosphate potassium buffer, pH 7.4

Working solution preparation:

Add 3-Cyano-7-ethoxycoumarin stock solution into incubation medium to a final concentration at 30 μM.

Protocol for assay:

1. Incubate cells cultured in 24-well plates with 30 μM 3-Cyano-7-ethoxycoumarin in 300 μL incubation medium for 90 min at 37°C in dark.

2. Collect the incubation medium and mark the tube.

3. Hydrolyzed the potential metabolite conjugates by incubation of supernatants with β-glucuronidase/arylsulfatase (150 Fishman units/ml and 1200 Roy units/ml, respectively) for 2 h at 37°C.

4. Dilute (1:2) the solution wit

3-Cyano-7-ethoxycoumarin is a cytochrome P-450 substrate. It can be dealkylated and generated 3-cyano-7-hydroxycoumarin, which is fluorescent at neutral pH values (excitation and emission wavelength maxima: 408 and 450 nm. respectively).

[1] White IN. A continuous fluorometric assay for cytochrome P-450-dependent mixed function oxidases using 3-cyano-7-ethoxycoumarin. Anal Biochem. 1988 Aug 1;172(2):304-10. doi: 10.1016/0003-2697(88)90449-6. PMID: 3189781.