With ∼1 × 10 7 genome equivalent copies of HBV, 1 × 10 5 cells were inoculated, or indicated otherwise, in the presence of ∼4% PEG8000 as reported for primary human hepatocyte and HepaRG cell (Gripon et al., 1993; Gripon et al., 2002; Schulze et al., 2007) . The cells were maintained subsequently in PMM and the medium was changed every 2-3 days. For immunofluorescence microscopy analysis, HBV-infected cells, with or without replating on glass coverslips for imaging, were fixed with 4% Paraformaldehyde (PFA) and permeabilized with 0.5% TritionX-100, and then stained either with 10 μg/ml 17B9 against HBsAg followed by FITC-labeled goat anti-mouse IgG, or with 5 μg/ml 1C10 against HBcAg followed by Qdot 655 VIVID donkey anti-mouse IgG. 1 μg/ml of DAPI was added to stain the nucleus before analyzing. The cell images were captured with a Nikon Eclipse Ti Fluorescence Microscope or a Zeiss LSM 510 Meta Confocal Microscope. Secreted viral antigens and intracellular viral replication intermediates cccDNA and/or RNAs were examined on indicated days after infection.