Bilirubin assay kit

ELISA assays were performed per manufacturer instructions. The MesoScale Discovery Multiplex (Rockville, MD) was used for initial analysis and findings were confirmed with BD OptEIA ELISA kits. Bilirubin Assay Kit (Sigma-Aldrich, St Louis, MO) and Alanine Aminotransferase 1 ELISA Kit (Biovision, Milpitas, CA) were used per manufacturer instructions.

HEK293 cells were transfected and cultured as described above in 6-well polystyrene-coated plates. Before analysis, the cells were washed and resuspended in 1 ml of 1× PBS and transferred to a 1.5 ml microfuge tube and pelleted at 400g 4 °C for 4 min. Supernatant was decanted and the cells were resuspended in 1 ml 1× PBS and counted using a TC20 Automated Cell Counter from BioRad. 1 × 10⁶ cells were pelleted and resuspended in Lysis Buffer (10 mM NaPi 50 mM NaCl, 1% v/v Triton X-100, 5 mM EDTA, 1 mM PMSF, and 1× Protease Arrest (G-Bioscience 786-437)), then lysed by freeze-thawing using 3 cycles of 10 min at −80 °C and 15 min at RT. Lysates were clarified by centrifugation at 21,100g in a table-top microfuge at 4 °C for 10 min 1% v/v DMSO was added to the cell lysate to solubilize the bilirubin. Total bilirubin was quantified using the Bilirubin Assay kit from Sigma-Aldrich exactly as described in the manufacturer’s protocol (MAK126). A standard curve was generated from bilirubin standards made up in cell lysis buffer with 1% v/v DMSO. Bilirubin concentrations were normalized to cell number, and a cellular concentration was determined by assuming a cell volume of 1.2 pl (109 (link)).

Plasma bilirubin levels were measured following Bilirubin Assay Kit protocol (Sigma-Aldrich, St. Louis, MO, USA) using 50 μL of blood plasma in each well of 96 well plates to determine the total bilirubin, direct bilirubin and blank absorbance. Absorbance was measured at 530 nm. The indirect bilirubin levels were determined by subtracting the total bilirubin numbers from the direct bilirubin, which is also known as conjugated bilirubin.