Incubation conditions for routine assessment of FmCYP3A4/5 of NCE

Culture Plate48-Well
Culture mediumWilliams’ E medium without phenol red, containing 2 mM glutamine and 15 mM Hepes
Incubation37°C in 5% CO2/95% air, humidified atmosphere under agitation (450 rpm)
Hepatocytes suspension prewarming30 min at 2 million/ml in similar conditions as incubation
Final incubation volume500 µl
Final hepatocyte concentration1 million/ml
Substrate concentration0.5 µM
Azamulin concentration3 µM
Final organic solvent concentration1% ACN
Incubation time4 h
Sampling time0, 30, 60, 120, 180, and 240 min
Sampling volume50 µl/time point