3 3 5 5 tetramethylbenzidine (tmb)

Culture supernatants were assayed for albumin as a measure of hepatocyte function using an enzyme-linked immunosorbent assay with horseradish peroxidase detection and 3,3',5,5'tetramethylbenzidine (TMB; Rockland Immunochemicals, Boyertown, PA, USA) as the substrate (Khetani and Bhatia, 2008) (link). Urea concentration in the supernatants was assayed using a colorimetric end point assay utilizing diacetyl monoxime with acid and heat (Stanbio Labs, Boerne, TX, USA) (Khetani and Bhatia, 2008) (link). Absorbance (520 nm) was measured on a Synergy H1 multimode plate reader (BioTek, Winooski, VT, USA). Initial CYP3A4 enzyme activities were initially measured by incubating the cultures with 3 µM luciferin-IPA substrate (Promega Life Sciences, Madison, WI, USA) for 1 hour. The luciferin metabolite was relatively quantified via luminescence detection on a Synergy H1 multimode plate reader according to the manufacturer's protocols. Viability of the cultures was quantified via the PrestoBlue TM assay (Thermo Fisher) per the manufacturer's instructions. Briefly, PrestoBlue substrate was combined with culture medium at a 1:9 ratio (v/v). The media containing the substrate was added to the cultures and incubated for 4 hours, and the metabolite (resazurin) was quantified via fluorescence detection (excitation/emission 560:590 nm) on the Synergy H1 multimode plate reader.