Quantifying Hepatitis C Virus Titers

12x10³/well Huh7.5 were seeded on 10mm diameter glass coverslip placed in 48-well plate.10-fold serially diluted viral stock was added to Huh7.5 cells in medium that was changed after 6 h. At 72 h post-infection, immunostaining against HCV core protein was performed. Briefly, cells were washed three times with PBS and fixed with ice-cold methanol at -20°C for 20 min. Cells were washed for three times with PBS, blocked for 1 h with PBS, 5%Normal Goat Serum (Biogenex, Fremont, CA, USA), 2% BSA and incubated with mouse anti-HCV Core (clone [C7-50], Abcam, Cambridge, UK) [81 (link)] diluted in blocking buffer 1:300 for an overnight at 4°C in a humidified chamber. Cells were washed with PBS and incubated for 1h at room temperature with secondary antibody goat anti-mouse IgG-Alexa568 (Invitrogen). The stained biopsy sections were mounted using SlowFade Gold Antifade reagent (Invitrogen) including DAPI for the nuclear counterstaining and stored in the dark. Images were acquired with confocal microscope (TCS SP5 II, Leica). The number of foci formed at the highest dilution was used to calculate the virus titer, which was expressed as the number of focus-forming units per milliliter of supernatant (FFU/ml). The titers of our JFH1 viral stock were usually in the range of 10⁴ to 10⁶ FFU/ml.