P450 glo cyp1a2 assay

Cytochrome P450 1A2 and 3A4 enzyme activity was evaluated in 24 well plates directly by testing luciferase activity with the P450-Glo CYP1A2 assay (V8422; Promega, Madison, WI, USA) and CYP3A4 assay (V9002; Promega). Briefly, cells in 1 ml microcapsules were incubated at 37°C in Krebs–Henseleit buffer containing Luciferin-1A2 or fresh medium containing Luciferin-IPA for 1 h. Then 50 μl buffer or culture medium was removed from each well and transferred to a 96-well opaque white plate and mixed with 50 μl luciferin detection reagent. After incubation for 20 min at room temperature, luminescence was measured using a microplate reader (DTX880; Beckman Coulter).

The P450 mediated detoxification was examined by Pentoxyresorufin O‐dealkylase assay, and the induction of CYP1A2 in monocytes and NeoHep was estimated using the P450‐Glo CYP1A2 assay (Promega, Madison, WI,
http://www.promega.com). Microsomes from NeoHep were isolated, and a P450‐Glo CYP3A4‐pentafluoro‐benzyl ether (PFBE) induction/inhibition assay (Promega) was performed. Human albumin and human clotting factor VII activity were detected by using commercial enzyme‐linked immunosorbent assay (ELISA) kits (KOMA BIOTECH, Seoul, Korea,
http://www.komabiotech.com) and a factor VII chromogenic activity assay kit (Assaypro, St. Charles, MO,
http://www.assaypro.com), respectively. Assays were performed according to the manufacturer's protocol, and detailed steps are given in the
supplemental online data.