IFN-α Inhibition Assay in vitro

Materials and Reagents

1. Mus dunni fibroblast cells
   
2. Friend murine leukemia virus (F-MuLV) (titer was measured as previously described (Robertson et al., 1991))
   
3. 3-Amino-9-ethylcarbazole (AEC) (Sigma-Aldrich, catalog number: A6926-100TAB )
   
4. Antibody 720 (mouse antibody against FV envelope protein), hybridoma supernatant (Robertson et al., 1991)
   
5. Bovine serum albumin (BSA) (PAA Laboratories GmbH, catalog number: K41-001 )
   
6. Ethanol 96% (Roth North America, catalog number: 5054.5 )
   
7. Superior FBS (fetal bovine serum, not heat-inactivated) (Biochrom, catalog number: S0615 )
   
8. Goat anti-mouse HRP (Dako, catalog number: P0477 )
   
9. Hydrogen peroxide 30% (Applichem, catalog number: A1134,0250)
   
10. N,N-dimethylformamide (Merck Millipore, catalog number: 1.03053.1000 )
    
11. PBS (GIBCO, catalog number: 14190-136 )
    
12. Penicillin/streptomycin (PAA Laboratories GmbH, catalog number: P11-010 )
    
13. Polybrene/Hexadimethrine bromide (Sigma-Aldrich, catalog number: H9268 )
    
14. RPMI 1640 (PAA Laboratories GmbH, catalog number: E15-840 )
    
15. Sodium Acetate (Merck Millipore, catalog number: 1062811000 )
    
16. Murine IFN-α (PBL, catalog number: 12100-1 )
    
17. Medium (see Recipes)
    
18. Washing Buffer (see Recipes)
    
19. 3-Amino-9-ethylcarbazole (AEC) substrate solution (see Recipes)
    

Equipment

1. Incubator (37 °C; 5% CO₂)
2. 24 well cell culture plate (Greiner Bio-one, catalog number: 662160 )
   

Procedure

Day 1:

1. Seed 7.5 x 10³ Mus dunni fibroblast cells in 500 μl media per well in 24-well plates.
   
2. Add IFN-α in increasing concentrations to the cells (use concentrations between 50 pg/ml to 10,000 pg/ml).
   
3. Controls: Without IFN-α; without virus.
   
4. Incubate the cells for 24 h at 37 °C (5% CO₂).
   
   

Day 2:

1. Decant media.
   
2. Add 1 ml fresh media supplemented with polybrene (8 μg/ml) to increase the infection efficiency.
   
3. Add 50 FFU (focus-forming units) F-MuLV to the wells.
   
4. Incubate for 3 days.
   
   

Day 5:

1. Decant media.
   
2. Fix cells with 500 μl 96% ethanol for 5 min at room temperature (RT).
   
3. Wash wells twice with 500 μl washing buffer (PBS + 0.1% BSA).
   
4. Add 250 μl supernatant of antibody 720 (mAB against FVenv) per well for 2 h.
   
5. Wash twice with 500 μl washing buffer.
   
6. Add 250 μl 2^nd antibody (goat anti-mouse HRP) to wells (diluted 1 to 500 in PBS).
   
7. Incubate for 1 h at RT.
   
8. Wash twice with 500 μl washing buffer.
   
9. Freshly prepare AEC substrate solution as indicated in Recipes section.
   
10. Add 250 μl substrate solution per well.
    
11. Incubate for 10-15 min at RT in the dark.
    
12. Decant supernatant in special waste container for toxic solvents.
    
13. Wash with 500 μl water.
    
14. Dry plates overnight.
    
    

Day 6:

1. Count foci
   Treatment with IFN-α should significantly decrease the numbers of foci compared to unstimulated cells (Figure 1).
   
   
   Figure 1. Representative example of Interferon-α Inhibition assay with Interferon-α (left picture without foci) and without Interferon-α (right picture with foci)

Recipes

1. Medium
   RPMI 1640
   10% FCS
   1% penicillin/streptomycin
   
2. Washing buffer
   PBS + 0.1% BSA
   
3. AEC substrate solution
   Dissolve 1 tablet AEC in 2.5 ml of N, N-dimethylformamide. Add 2.5 ml of the substrate solution to 47.5 ml of 50 mM sodium acetate buffer, pH 5.0. Add 25 μl of fresh 30% hydrogen peroxide immediately prior to use.
   

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (GRK 1045).

References

1. Dittmer, U., Brooks, D. M. and Hasenkrug, K. J. (1998). Characterization of a live-attenuated retroviral vaccine demonstrates protection via immune mechanisms. J Virol 72(8): 6554-6558. 
2. Gibbert, K., Joedicke, J. J., Meryk, A., Trilling, M., Francois, S., Duppach, J., Kraft, A., Lang, K. S. and Dittmer, U. (2012). Interferon-alpha subtype 11 activates NK cells and enables control of retroviral infection. PLoS Pathog 8(8): e1002868.
3. Gerlach, N., Gibbert, K., Alter, C., Nair, S., Zelinskyy, G., James, C. M. and Dittmer, U. (2009). Anti-retroviral effects of type I IFN subtypes in vivo. Eur J Immunol 39(1): 136-146.
   
4. Robertson, M. N., Miyazawa, M., Mori, S., Caughey, B., Evans, L. H., Hayes, S. F. and Chesebro, B. (1991). Production of monoclonal antibodies reactive with a denatured form of the Friend murine leukemia virus gp70 envelope protein: use in a focal infectivity assay, immunohistochemical studies, electron microscopy and western blotting. J Virol Methods 34(3): 255-271.