Materials and Reagents

1. Cell line: HCV-replicon Huh-7 human hepatoma cell line (HCV genotype 1b subgenomic replicon pFKI389neo/NS3–3’/5.1 containing the neomycin resistant gene, provided by R. Bartenschlager, Heidelberg University, German) (Lohmann et al., 1999; Krieger et al., 2001)
   
2. 0.1% Trypsin-EDTA (WELGENE, catalog number: LS015-01 )
   
3. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, Hyclone^TM, catalog number: SH30919.03 )
   
4. 100x Penicillin/Streptomycin solution (Thermo Fisher Scientific, Hyclone^TM, catalog number: 3V30010 )
   
5. Lipofectamine 2000 (Life Technology, catalog number: 11668-019 )
   
6. NaCl (Sigma-Aldrich, catalog number: S5886 )
   
7. KCl (Sigma-Aldrich, catalog number: P5405 )
   
8. Na₂HPO₄ (Sigma-Aldrich, catalog number: S3264 )
   
9. KH₂PO₄ (Sigma-Aldrich, catalog number: P9791 )
   
10. NaOH (Sigma-Aldrich, catalog number: S5881 )
    
11. 1x Phosphate Buffered Saline (PBS) (see Recipes)
    
12. Complete Dulbecco’s modified Eagle medium with high glucose (DMEM) (Thermo Fisher Scientific, Hyclone^TM, catalog number: SH30243.01 ) (see Recipes)
    
13. 50 mg/ml G418 (Merck KGaA, catalog number: 345810 ) (see Recipes)
    
14. 2% Paraformaldehyde solution (Sigma-Aldrich, catalog number: 158127 ) (see Recipes)
    
15. 1% Methylene blue (Duksan Scientific, catalog number: MEE0-22002 ) (see Recipes)
    

Equipment

1. 35 mm cell culture plate (Corning, catalog number: 430165 )
   
2. 100 mm cell culture plate (BD Bioscience, catalog number: 353003 )
   
3. 5% CO₂, 37 °C Incubator (Thermo Fisher Scientific, catalog number: 311 )
   

Procedure