Materials and Reagents

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

Equipment

Procedure

Day 1:

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

Day 2:

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

Day 5:

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

Day 6:

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

Medium
RPMI 1640
10% FCS
1% penicillin/streptomycin
Washing buffer
PBS + 0.1% BSA
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

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.
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.
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.
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.