(A-C) Huh7.5 cells seeded in poly-D-lysine coated 96-well plates the previous day, were incubated with (A) SA13(5a) and sf-SA13(5a) diluted in DMEM + 10% FBS (black bars) or AEM (grey bars), (B) sf-SA13(5a) diluted in different media with supplements as indicated or (C) sf-SA13(5a) diluted in AEM supplemented with different concentrations of FBS. % FBS in growth medium indicates the final FBS concentration. (A–C) Cells were incubated with virus mixes for 3 hours. After incubation, fresh DMEM + 10% FBS was added to all wells. Cells were incubated for 48 hours before they were fixed, stained and the number of single HCV NS5A positive cells per well was determined by automated counting as described in Materials and Methods. Error bars represent SEM of triplicates. For (B), the mean infectivity (HCV NS5A positive cells/well) of triplicate wells of the reference culture (DMEM + 10% FBS, black bar) was set to 100%. The number of HCV NS5A positive cells / experimental well was related to this mean to calculate % infectivity relative to the reference culture.