Human albumin enzyme linked immunosorbent assay

Albumin and urea production as well as lactate dehydrogenase (LDH) release were measured before the drug-metabolism study (4 days postseeding). Albumin production was measured in supernatant using a human albumin enzyme-linked immunosorbent assay (Assay Pro, St. Charles, MO). Urea was quantified with a colorimetric assay kit (BioAssay Systems, Hayward, CA) and LDH secretion was measured using the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Southampton, UK). Albumin, urea, and LDH were also measured postdose at the end of the drug-metabolism study (day 5 for wells treated with phenacetin; day 6 for wells treated with diclofenac, propranolol, lidocaine, and ibuprofen; and day 7 for wells treated with prednisolone). At the end of the experiment, the scaffolds/tissues were removed and washed with phosphate-buffered saline. Bright field images were taken using an inverted light microscope (Leica, Milton Kaynes, UK).

Albumin secretion was measured in culture supernatant using a human albumin enzyme-linked immunosorbent assay (Assay Pro, United States). Urea was quantified with a colorimetric assay kit (BioAssay Systems, United States) and lactate dehydrogenase (LDH) activity was measured using the CytoTox 96^® Non-radioactive cytotoxicity assay (Promega, United Kingdom). Aspartate transaminase (AST) and alanine aminotransferase (ALT) clinical chemistry assays were performed at the College of American Pathologists certified clinical laboratories in the University of Pittsburgh Medical Center (Pittsburgh, United States). Total glutathione was quantified using the GSH-Glo™ Glutathione Assay (Promega, United Kingdom). Microtissues were lysed from scaffolds in glutathione lysis buffer and each lysate was diluted 100-fold in fresh lysis buffer before luminescent quantification of total glutathione against a standard curve according to vendor instructions. The glutathione content was normalised to total cellular protein. Mitochondrial activity was assessed using Cell Proliferation Reagent WST-1 (Roche, United Kingdom) at a 20-fold dilution in cell culture medium.

Albumin secretion was measured with a human albumin enzyme-linked immunosorbent assay (Assaypro, St. Charles, MO). Lactate dehydrogenase (LDH) activity was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Southampton, UK).