P450 glo cyp1a2

CYP1A2, CYP2B6 and CYP3A4 activity was measured by using P450-Glo™ CYP1A2 (#V8421), CYP2B6 (#V8321) and CYP3A4 (#V9001) Assay systems (Promega) according to manufacturer’s protocols. Results were calculated against the number of viable cells in the culture which was determined by using CellTiter-Glo 3D Cell Viability Assay (Promega) according to manufacturer’s instructions.

Dimethyl sulfoxide (DMSO) was provided by Glentham Life Sciences (Corsham, UK). PCR-grade nano-pure water was purchased from Jena Bioscience (Munich, Germany). Vivid CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 screening kits, EOMCC, and BOMCC were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Betanin, bupropion, furafylline, omeprazole, quinidine, rifampicin, and Krebs-Henseleit buffer were supplied by Sigma-Aldrich (St. Louis, MO, USA). Sulfaphenazole, fluvoxamine, and ketoconazole were obtained from Toronto Research Chemicals (Toronto, ON, Canada). P450-Glo CYP1A2, P450-Glo CYP2B6, and P450-Glo CYP2C9 assay kits were provided by Promega (Wisconsin, MD, USA).

Following the manufacturer's protocol, cytochrome P450 enzyme activity was measured using a P450-Glo Assay Kit (Promega, Madison, WI, USA). Approximately 100 aggregates were isolated from each group of culture systems at the end of the hepatic differentiation. For the CYP3A4 assay, the aggregates were incubated in an hepatocyte culture medium (HCM) with BulletKit supplementation and 20 μM rifampicin solution (Sigma-Aldrich) for 48 h. CYP1A2 activity was evaluated by incubating the aggregates in the HCM Hepatocyte Culture Medium with BulletKit containing 50 μM omeprazole solution (Sigma-Aldrich) for 48 h. For the CYP2B6 activity assay, the aggregates were incubated with the HCM with BulletKit supplement containing 1000 μM phenobarbital solution (Sigma-Aldrich, USA) for 48 h. The CYP activity value was normalized to the number of cells tested. The activity of CYP2B6 was tested using P450-Glo CYP2B6 (Promega, Madison, WI, USA), CYP3A4 using P450-Glo CYP3A4 (Promega), and CYP1A2 using P450-Glo CYP1A2 (Promega).