Luciferin ipa

Albumin was measured using a sandwich-based enzyme linked immunosorbent assay (ELISA, Bethyl Laboratories, Montgomery, TX) with horseradish peroxidase detection and 3,3’5,5’-tetramethylbenzidine substrate (TMB, Rockland Immunochemicals, Boyertown, PA). Urea concentration was measured from collected supernatants using diacetyl monoxime with acid and heat (Stanbio Labs, Boerne, TX).^{[48 (link)]} Absorbance of samples was read on the Synergy H1 multimode plate reader (Biotech, Winooski, VT).
CYP3A4 and CYP2C9 enzyme activities were measured after the incubation of cultures for 1 hour with luciferin-IPA (Promega Life Sciences, Madison, WI) or for 3 hours with luciferin-H (Promega), respectively, followed by the processing of collected supernatants per the manufacturer’s recommendations; luminescence was quantified with the Synergy H1 multimode reader. CYP1A2 and CYP2A6 enzymatic activities were measured by incubating cultures for 1 hour with 50 µM coumarin (Sigma-Aldrich) or for 3 hours with 5 µM 7-ethoxyresorufin (Sigma-Aldrich), respectively. The CYP2A6-generated metabolite, 7-hydroxycoumarin (7-HC), and CYP1A2-generated metabolite, resorufin, in culture supernatants were quantified using fluorescence measurements (excitation/emission 355/460 nm for 7-HC and 550/585 nm for resorufin) on the Synergy H1 multimode reader.

Cell adhesion was assessed via double-stranded DNA (dsDNA) content in adherent cells. DNA concentration was measured using an AccuBlue dsDNA quantitation kit (Biotium, Hayward, CA) or a Quant-iT PicoGreen dsDNA assay kit (Molecular Probes, Eugene, OR). Culture supernatants were assayed for albumin levels using a competitive enzyme-linked immunosorbent assay (ELISA, MP Biomedicals, Santa Ana, CA) with horseradish peroxidase detection and 3,3′,5,5′-tetramethylbenzidine (TMB, Rockland Immunochemicals, Boyertown, PA) as the substrate, as previously described.¹³ Urea concentration in culture supernatants was assessed using a colorimetric endpoint assay utilizing diacetyl monoxime with acid and heat (Stanbio Labs, Boerne, TX).¹³ CYP3A4 enzyme activity was measured by first incubating the cultures with substrate (luciferin-IPA from Promega Life Sciences, Madison, WI) for 1 hour at 37°C and then detecting the luminescence of the produced metabolite (luciferin) according to the manufacturer’s protocols. CYP2A6 enzyme activity was measured by incubating the cultures with coumarin for 1 hour at 37°C and measuring the fluorescent metabolite, 7-hydroxy-coumarin (Sigma-Aldrich, St. Louis, MO). Absorbance and luminescence for the aforementioned assays were measured using a BioTek (Winooski, VT) Synergy H1 multi-mode plate reader.