Fig. 1. Optimizing HCV infection of primary human hepatocytes. (a, b) Human hepatocytes were isolated according to previously published protocols (Mitry, 2009) and maintained in Williams E medium supplemented with 10 % FBS/5 mM HEPES/insulin/dexamethasone. Hepatocytes were plated at 4×10⁴ cm⁻² on collagen-coated plates and infected with HCVpp-H77 or VSV-Gpp prepared as previously described (Hsu et al., 2003). Briefly 293T HEK cells were transfected with two plasmids, one encoding the HIV provirus expressing luciferase and the other encoding HCV E1E2, VSV-G or a no-envelope control. Supernatants were collected 48 h later, clarified, evaluated for HIV p24 content and used to infect target cells for 8 h with an inoculum of 1 ng p24 per well at 1, 2 and 4 days post-seeding. Virus was removed by washing and cultures were maintained at 37 °C for 72 h. At 72 h post-infection, cells were lysed and luciferase activity was measured using a luminometer (Lumat LB 90507). Cells were incubated with anti-SR-BI or anti-CD81 (2s131) monoclonal antibodies at 5 µg ml⁻¹ prior to being inoculated with virus. Data are presented as relative light units (RLU) from which a no-envelope pseudotype control value has been subtracted, and are representative of three experiments. Error bars indicate sd from the mean (n = 3); ***P<0.001; ns denotes no statistical significance. (c) Cells were infected with HCVcc SA13/JFH as previously described (Jensen et al., 2008). Virus-containing medium (1.8×10⁶ focus-forming units ml⁻¹, approximate m.o.i. 0.01) was added to target cells for 8 h followed by washing and change of medium. Infection was realized 72 h later by quantitative reverse transcriptase PCR detection of the cellular viral RNA load using the Cells Direct kit (Life Technologies). Data are presented as HCV RNA copy number relative to GAPDH and are representative of three independent experiments. ***P<0.001. (d) Confocal microscopy images of HCV receptor molecules CD81 (2s131), claudin-1 and occludin (Life Technologies) in PHHs at 1, 2 or 4 days post-seeding on glass coverslips. Cells were methanol-fixed and permeabilized with saponin (Sigma) followed by incubation with respective antibodies (1 : 1000) for 1 h at room temperature in a PBS/BSA solution. Cells were washed three times before the addition of species-appropriate Alexa-594-conjugated secondary antibodies (1 : 1000) for 1 h at room temperature. After a further wash the nuclei were counterstained with DAPI and coverslips were mounted on glass slides using ProLong Gold antifade (Life Technologies). Scale bars represent 20 µm. (e) Western blot detection of HCV receptor molecules including SR-BI in PHHs lysed at 1, 2 or 4 days post-seeding using a previously published protocol (Farquhar et al., 2012).