HCV spread in standard monolayer cell culture was evaluated by immunostaining using primary antibody anti-NS5A-9E10 at 1:300017 (link), secondary antibody Alexa Flour® 488 goat anti-mouse IgG at 1:500 (Lifetechologies) and Hoechst 33342 at 1:1000 as described24 (link),65 (link). The percentage of infected cells was determined by fluorescence with a Zeiss Axio Vert.A1 microscope.
To determine HCV infectivity titers, Huh7.5 cells were plated at 6000 cells/well in poly-D-lysine 96-well plates (Thermo Scientific). The next day, serially diluted viral supernatant was added (minimal dilution was 2-fold), testing each dilution in triplicates, and incubated for 48 hours24 (link),66 (link). Cells were immunostained as described using anti-NS5A-9E10 at 1:500017 (link) and ECL sheep anti-mouse IgG horseradish-peroxidase linked whole antibody (GE Healthcare) at 1:500, followed by incubation with DAB substrate (DAKO). FFU were counted automatically with an ImmunoSpot Series 5 UV Analyzer (CTL Europe Gmbh) with customized software. Titers were calculated as described35 (link),67 (link).
HCV Core titers in culture supernatant were determined using the ARCHITECT HCV Ag assay (Abbott).
HCV RNA titers were determined from RNA extracted from 200 μL pre-diluted supernatant using the Total Nucleic Acid Isolation Kit (Roche Applied Science) and by Taqman real-time PCR as described65 (link).
To determine HCV infectivity titers, Huh7.5 cells were plated at 6000 cells/well in poly-D-lysine 96-well plates (Thermo Scientific). The next day, serially diluted viral supernatant was added (minimal dilution was 2-fold), testing each dilution in triplicates, and incubated for 48 hours24 (link),66 (link). Cells were immunostained as described using anti-NS5A-9E10 at 1:500017 (link) and ECL sheep anti-mouse IgG horseradish-peroxidase linked whole antibody (GE Healthcare) at 1:500, followed by incubation with DAB substrate (DAKO). FFU were counted automatically with an ImmunoSpot Series 5 UV Analyzer (CTL Europe Gmbh) with customized software. Titers were calculated as described35 (link),67 (link).
HCV Core titers in culture supernatant were determined using the ARCHITECT HCV Ag assay (Abbott).
HCV RNA titers were determined from RNA extracted from 200 μL pre-diluted supernatant using the Total Nucleic Acid Isolation Kit (Roche Applied Science) and by Taqman real-time PCR as described65 (link).