Human albumin elisa kit

Gene expression analysis by RNA sequencing (RNA-seq) and quantitative PCR is described in Supplementary Materials and methods. Albumin and urea secretion into the media was measured using a human albumin ELISA kit and the QuantiChrom™ urea assay kit (both from Bethyl Laboratories). Comparisons with fetal and adult hepatocyte data used the unpaired two-tailed Student’s t test. CYP3A activity was assessed in duplicate by incubation with P450-Glo™ CYP3A4 assay reagent (Luciferin-PFBE; Promega Ltd). For CYP analysis by mass spectrometry, cells were incubated with 1 mM testosterone or 1 mM dextromethorphan (Sigma, UK) in HCM. Conditioned medium was collected and diluted 1:1 in 0.5 μM phenacetin (Sigma) stop solution in methanol. CYP activity was calculated per min incubation. Alcohol dehydrogenase activity of cell lysates was assessed using a detection kit following the manufacturer’s instructions (Abcam, UK). Results were standardized to the amount of protein measured by Bradford assay.

To test the functionality of the hepatocytes and hepatoma cultures, aliquots of media was collected every 2 days from both static and dynamic perfusion cultures. Albumin content in the medium was measured using the Human Albumin ELISA kit (E88-129, Bethyl Laboratories, Inc. Montgomery, TX, USA) following manufacturer’s instructions. Culture media was diluted 1:2 before assaying.
Urea content was measured using colorimetric Urea assay kit (ab83362, Abcam, Cambridge, UK) following manufacturer’s instructions. Both static and dynamic perfusion culture media was diluted 1:32 before assaying.

NOD.Cg-Prkdc^scid Il2rg^tm1Sug Tg(Alb-UL23)7-2/ShiJic (TK-NOG) mice are a specially designed strain (43 (link)) that allows for the conditional depletion of mouse hepatocytes and engraftment of their human cell counterparts. We used these animals as previously described (12 (link)). Eight- to 10-week-old TK-NOG male mice were selected by genotyping and injected with ganciclovir (10 and 30 mg/kg) 7 and 5 days to achieve significant damage of mouse liver as evident by elevated to ALT levels (200 to 400 IU/ml) before transplantation of human hepatocytes. Human hepatocytes were obtained from Lonza (lot no. 4145; Walkersville, MD, USA). Two million hepatocytes were intrasplenically infused. The levels of hepatocyte engraftment were monitored starting from 1 month after transplantation. The chimerism rate correlated with serum hAlb levels (43 (link)) measured using the Human Albumin ELISA Kit (Bethyl Laboratories Inc., Montgomery, TX). At 2 months after transplantation, animals were intravenously infected with patient-derived sera samples containing ~10⁶ HBV DNA, and the drug treatment was started at 2 months after infection. Overall, the mice were followed for 3 months after drug injection. The duration of experimental animal life reached ~9 months.