Materials and Reagents

7AAD (7-amino-actinomycin D) (BD Pharmingen, catalog number: 51-68981E )
Bovine serum albumin (BSA) (PAA Laboratories GmbH, catalog number: K41-001 )
DMEM (Life Technologies, Gibco^®, catalog number: 41966-029 )
Superior FBS (fetal bovine serum, not heat-inactivated) (Biochrom, catalog number: S0615 )
Mx/RAGE7 cells (virus-transformed adherent cell line with a temperature-inducible promotor; must be cultured at 32 °C; cells express the Mx transgene and a promotorless eGFP gene which is expressed due to type I IFN stimulation ) (Bollati-Fogolin and Muller, 2005)
PBS (Life Technologies, Gibco^®, catalog number: 14190-136 )
Penicillin/streptomycin (PAA Laboratories GmbH, catalog number: P11-010 )
Propidium iodide (eBioscience, catalog number: 00-6990-50 )
Murine IFN-α (PBL, catalog number: 12100-1 )
Sodium azide (Applichem, catalog number: A1430.0010 )
Sodium pyruvate (Life Technologies, Gibco^®, catalog number: 11360-039 )
Trypsin EDTA (PAA Laboratories GmbH, catalog number: L11-004 )
β-mercaptoethanol (Life Technologies, Gibco^®, catalog number: 31350-010 )
Media for Mx/RAGE7 cells (see Recipes)
FACS buffer (see Recipes)

Equipment

96-well flat bottom plate (Falcon BD Labware, catalog number: 3072 )
1.5 ml microfuge tubes
FACS tubes (BD Biosciences, Falcon^®, catalog number: 352054 )
Flow cytometer (e.g. BD LSR II)
Incubator (37 °C; 5% CO₂)
Incubator (32 °C; 5% CO₂)

Procedure

Different murine IFN-α subtypes (IFN-α1, -α2, -α4, -α5, -α6, -α9, -α11) were produced as already described (Gerlach et al., 2009).

Day 1:

Seed Mx/RAGE7 cells in a 96 well cell culture plate (2 x 10⁴ cells per well in 200 μl medium).
Grow the cells for 24 h at 32 °C.

Day 2:

Perform serial dilutions (log10) of produced IFN-α subtypes in medium in 1.5 ml tubes.
Perform serial dilutions (log2) of recombinant IFN-α subtypes (PBL) with known concentrations from 1,000 U/ml to 31.25 U/ml (= standards) in 1.5 ml tubes.
Decant medium of Mx/RAGE7 cells.
Add 200 μl of the IFN-α solutions with known (standards) and unknown concentrations to the cells.
As negative control add 200 μl of medium without IFN-α.
Incubate the samples for 24 h at 37 °C.

Day 3:

Decant the medium.
Add 200 μl fresh medium to the cells.
Incubate the samples for 48 h at 37 °C.

Day 5:

Decant the medium.
Wash cells with 200 μl PBS.
Add 50 μl of trypsin EDTA (1x) 0.05% to the cells at room temperature until they suspend.
Harvest suspended cells in FACS tubes containing 1 ml of PBS.
Centrifuge cells (300 x g; 5 min).
Resuspend cells with 250 μl FACS buffer.
Add 2.5 μl 7AAD or 0.5 μl propidium iodide per sample to exclude dead cells.
Immediately analyze cells with flow cytometer.
IFN-α treated Mx/RAGE7 cells express eGFP (Figure 1).
Perform standard curve with samples treated with known IFN-α concentrations (graph the data for the standard curve (Figure 2), the IFN-α titer can be determined by comparison).
Calculate concentrations of unknown samples.

Figure 1. Representative dot plots of Mx/RAGE7 cells without IFN-α (upper panel) and with IFN-α (lower panel)

Figure 2. Standard curve of IFN-α
Recipes
1. Media for Mx/RAGE7 cells
  DMEM
  10% FBS
  1 mM sodium pyruvate
  1% penicillin/streptomycin
  50 μM β-mercaptoethanol
2. FACS buffer
  PBS
  0.1% BSA
  0.02% sodium azide
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (GRK 1045).
References
1. Bollati-Fogolin, M. and Muller, W. (2005). Virus free, cell-based assay for the quantification of murine type I interferons. J Immunol Methods 306(1-2): 169-175.
2. 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.
3. 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.