Luciferin me

Luciferin-H, Luciferin-ME, Luciferin-1A2, and the NADPH regeneration system were all obtained from Promega (Madison, WI, USA); diclofenac and DMSO were from Sigma Aldrich (St. Louis, MO, USA); nicotine was from Xi’an Taima Biological Engineering (Xi’an, China); Luciferin-BE, Luciferin-2FBE, Luciferin-3FBE, and Luciferin-4FBE were synthesized in-house using a previously published protocol [12 (link)]; 1-benzylimidazole was from Alfa Aesar (Shanghai, China); ketoconazole and letrozole were from Dingguo Biotechnology (Beijing, China); Triton-X100 was from Leagene (Beijing, China); glycerol was from Dingguo (Tianjin, China); Tris-HCl was from AKZ-Biotech (Tianjin, China); potassium chloride, ammonium bicarbonate, potassium dihydrogen phosphate, and potassium hydrogen phosphate were from Jiangtian Chemical (Tianjin, China); ethyl acetate of analytical grade was from Yuanli Chemical (Tianjin, China); and white 96-well microtiter plates were from Nunc (Thermo Fisher Scientific, Langenselbold, Germany). All other chemicals and reagents used were of the highest grade available.

To measure Cyp3A and Cyp1A2 activity, 50 μM of Luciferin-PFBE substrate (Promega) or 100 μM of Luciferin-ME (Promega) were incubated with liver spheres maintained in liver sphere medium. Cytochrome P450 activity was measured 24 h later using the P450-Glo assay kit (Promega) according to manufacturer’s instructions. Data were normalised by total protein content measured using bicinchoninic acid (BCA) assay (Thermo Fisher).