P450 glo cyp1a2 assay system

CYP1A2 enzyme activity in primary mouse Hep was analyzed using the P450-Glo™ CYP1A2 assay system (Promega, Madison, WI) with Luciferin-ME as substrate, according to the manufacturer's protocol. The results were analyzed using a GloMax^® 96 Microplate Luminometer (Promega).

For glycogen storage analysis, Periodic acid–Schiff (Muto Pure Chemical, Tokyo, Japan) staining was performed on paraffin slides of iHLOs or iHEAs following the manufacturer’s instructions. For low-density lipoprotein (LDL) uptake assays, DiI-ac-LDL (Biomedical Technologies, Massachusetts, USA) was added the culture medium (200 ng/mL) and incubated for 4 h at 37 °C and 5% CO₂. After washing with PBS three times, cells were imaged by fluorescent microscopy (Leica, Wetzlar, Hesse, Germany) for LDL uptake analysis. For CYP P450 activity assays, MEFs, 2D-iHs, 2D-iHEs, iHLOs, and iHEAs were placed in an iHEA medium without dexamethasone supplement. We treated 2 μM of 3-methylcholanthrene (3-MC) CYP inducers for 72 h. The fresh medium containing 3-MC was replaced every 24 h. Dimethyl sulfoxide (DMSO) was used as a control. The P450-Glo CYP1A2 assay system (Promega, Madison, WI, USA) was used according to a modified manufacturer’s protocol. The luminescence was measured with a GLOMAX 96 micrometer luminometer (Promega, Madison, WI, USA). For the quantification of albumin secretion and urea production, the amounts of albumin and urea in the culture media were analyzed using the albumin ELISA Kit (Bethyl Laboratories, Waltham, MA, USA) and urea kit (BioAssay systems, Hayward, CA, USA), respectively, according to the manufacturer’s instructions.