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HCVpp production

JC Joan Capella-Pujol
MG Marlon de Gast
LR Laura Radić
IZ Ian Zon
AC Ana Chumbe
SK Sylvie Koekkoek
WO Wouter Olijhoek
JS Janke Schinkel
MG Marit J. van Gils
RS Rogier W. Sanders
KS Kwinten Sliepen
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HCV pseudoparticle (HCVpp) generation and neutralization assays were performed as described elsewhere105. One day prior to transfection for generating HCVpp, 1.5 × 106 HEK293T or HEK293TCD81KO cells were seeded on a 10 cm2 dish. Cells were co-transfected with three plasmids: MLV Gag-Pol packaging construct, firefly luciferase106 and E1E2 in optimized ratios105 with a total amount of 6 µg of DNA and 12 µl of Lipofectamine 2000 (Invitrogen) in Opti-MEM (ThermoFisher). After an incubation overnight, Opti-MEM was replaced by DMEM (Gibco)/10% fetal bovine serum (FCS)/0.1% Penicillin-Streptomycin (PS). Two days later, the supernatant containing the HCVpps was passed through a 0.45 µm filter and frozen at −80 °C for long-term storage or 4 °C when used within a week.

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