Collagen coated 6 well plates

Cryo-preserved primary human hepatocytes were obtained from Zenotech (Kansas City, KS). Cells were thawed with OptiThaw medium and enumerated with OptiCount medium in a standard hemacytometer. Cells were diluted to a final concentration of 10⁶ cells/ml of OptiPlate medium. Cells were plated in collagen-coated 6 well plates (BD Biosciences) at 1 ml per well. After 4 hours of plating, the medium was changed to OptiCulture medium for the duration of culture. Medium was exchanged every 24 hours.

Hoechst 33342 (Thermo, Grand Island, NY) was used to assess compacted nuclear chromatin and apoptosis. Hepatocytes were isolated from eight-week-old male WT mice and miR-150 KO mice. The isolated hepatocytes were plated onto collagen-coated 6-well plates (BD Biosciences, San Jose, CA) at a density of 1×10⁶ cells per well. After 2 hours of incubation to allow attachment, the hepatocytes were exposed to the medium with or without Jo2 0.5 μg/mL plus CHX 10 μg/mL for 4 hours. Following washing with PBS, the cells were incubated with Hoechst 33342 (1 μg/mL in 1× DPBS) for 30 minutes at room temperature in the dark. The nuclear morphology was observed at 200× under fluorescence microscope using a wavelength at 460 to 490 nm.

Freshly isolated PTH were seeded into collagen-coated 6-well plates (BD Biosciences, San Jose, CA) and maintained in Hepato-Stim Hepatocyte Defined medium (HDM) (BD Biosciences) at 5 × 10⁵ cells per well. Twenty-four hours post-seeding, PTH were infected with concentrated viruses prepared from HepAD38 supernatants (genotype D) at a multiplicity of infection (MOI) of 100 GE per cell. For peptide-mediated inhibition, viruses were pre-incubated with 4B10 (0.01 nM, 0.1 nM, 1 nM, 10 nM or 100 nM) or LA-20 (100 nM) for 30 min at 37 °C in HDM prior to infection. After incubation at 37 °C overnight, cells were washed 5 times with PBS and cultivated for 12 days. For HBV infection of PHH, freshly prepared PHH were seeded into collagen-coated wells in supplemented WEM at 5 × 10⁵ cells per well. Sera from HBV-positive patients (genotypes B and C) were pooled and pre-incubated with 4B10 (0.05 nM, 0.5 nM, 5 nM, 50 nM or 500 nM) or LA-20 for 30 min at 37 °C in WEM pre-mixed with 2% DMSO and 4% PEG 8000, and added three days post-seeding at an estimated MOI of 400. PTH and PHH culture media were changed every two days, and hepatitis B surface antigen (HBsAg) and e antigen (HBeAg) in collected supernatants were determined using enzyme linked immunosorbent assay (ELISA) kits (Kehua). All infections were repeated at least twice.