IFN-α Inhibition Assay in vitro    

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Original research article

A brief version of this protocol appeared in:
PLOS Pathogens
Aug 2012


During viral infections Interferon-α (IFN-α) is expressed by infected host cells. IFN-α binds to its receptor (IFNAR1/2), which leads to the activation of downstream signaling via JAK-STAT. This signaling cascade results in the expression of several hundred different genes, so called interferon-stimulated gene, which lead to an antiviral state of the infected and the neighboring cells.

Keywords: Type I IFNs, IFN-alpha subtypes, Friend Murine leukemia virus, Antiviral effect

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)


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


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 2nd 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)


  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.


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


  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.
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gibbert, K. (2013). IFN-α Inhibition Assay in vitro. Bio-protocol 3(12): e802. DOI: 10.21769/BioProtoc.802.
  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.

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