Cytochrome P450 Inhibition, Fluorescence Detection (IC50 Determination)
Assay System: 
  • Individual human recombinant CYP isoforms expressed in baculovirus-infected insect cells.
  • Inhibition potential of test compound against recombinant CYP isoforms measured at different compound concentrations. 
  • Reversible inhibition is determined by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with fluorescence detection.

Assay Conditions:

Test Compound Concentration   Eight concentrations ranging from 0.05 µM to 100 µM
Percent Organic Solvent in Incubation   2% acetonitrile
CYP Isoforms Available for Testing   CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4
Incubation Time   15-45 minutes (depending on CYP isoform)
pH   7.4
Temperature   37 °C
Analysis   Fluorescence Detection
Number of Repeats   2

Deliverable: IC50 of test compound and positive control compound.

Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.


Probe Substrate
3-Cyano-7-ethoxycoumarin
7-Ethoxy-4-trifluoromethylcoumarin
Dibenzylfluorescein 
7-Methoxy-4-trifluoromethylcoumarin
3-Cyano-7-ethoxycoumarin
3-[2-(N,N-diethyl-N methylamino)ethyl]-7-methoxy-4-methylcoumarin
7-Benzyloxy-4-(trifluoromethyl)-coumarin
CYP Isoform
1A2
2B6
2C8
2C9
2C19
2D6
3A4
Positive Control Inhibitor
Furafylline
Tranylcypromine
Quercetin
Sulfaphenazole
Tranylcypromine
Quinidine
Ketoconazole