Measurement of Type I Interferon Production

Rosalind E. Seeds1, Joanna L. Miller1

1 Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 14.21
DOI:  10.1002/0471142735.im1421s92
Online Posting Date:  February, 2011
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The Basic Protocol in this unit describes measurement of murine interferon (IFN)α/β by intracellular staining for these cytokines and detection by flow cytometry. Alternate protocols detail an enzyme‐linked immunoabsorbent assay (ELISA) for IFNα and a biological assay to measure IFN. The FACS assay allows measurement of IFNα/β production by defined cell populations, while ELISA measures secreted IFNα. The bioassay measures functional antiviral activity and is the most sensitive of the assays discussed. These assays therefore provide complementary methods to assess IFN production by murine cells. Curr. Protoc. Immunol. 92:14.21.1‐14.21.11. © 2011 by John Wiley & Sons, Inc.

Keywords: intracellular cytokine staining; ELISA; bioassay; type I interferon; cytokines

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Table of Contents

  • Introduction
  • Basic Protocol 1: Intracellular Staining for IFNα and IFNβ
  • Alternate Protocol 1: Measurement of IFNα by ELISA
  • Alternate Protocol 2: Measurement of IFN by Bioassay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Intracellular Staining for IFNα and IFNβ

  • Single cell suspension of splenocytes (prepared as described in Seeds et al., )
  • RPMI medium (see recipe for serum‐free RPMI) supplemented with 10% (v/v) heat‐inactivated fetal bovine serum
  • CpG ODN 2216 (InvivoGen) or HAU β‐propiolactone or ultraviolet‐inactivated influenza virus
  • GolgiPlug or Brefeldin A (BD Biosciences)
  • PBS ( appendix 2A)
  • 1% paraformaldehyde in PBS
  • FACS block solution (see recipe)
  • Anti‐pDC monoclonal antibody (mAb) such as 120G8 (Asselin‐Paturel et al., ) directly labeled with FITC or Alexafluor488 (IMGENEX)
  • Biotinylated anti‐IFNα mAb, such as clones 4EA1 (Kawade and Watanabe, ) or RMMA‐1 (PBL InterferonSource)
  • Biotinylated anti‐IFNβ mAb, such as clone 7FD3 (Kawade and Watanabe, ) or RMMB‐1 (PBL Interferon source)
  • Appropriate isotype control mAb
  • FACS wash solution (see recipe), ice cold
  • Streptavidin allophycocyanin (SA‐APC) or other appropriate secondary detection reagent
  • Class II biological safety cabinet
  • 6‐ or 24‐well flat‐bottomed tissue culture plates
  • 1.5‐ml microcentrifuge tubes
  • 96‐well V‐bottomed plates
  • 37°C, 5% CO 2 humidified incubator
  • Cell scrapers (BD Falcon)
  • Refrigerated centrifuge
  • Flow cytometer with minimum ability to measure two colors

Alternate Protocol 1: Measurement of IFNα by ELISA

  • ELISA wash solution (see recipe)
  • ELISA blocking solution (see recipe)
  • Murine IFNα standard (PBL InterferonSource)
  • Rabbit polyclonal Ab against IFNα (PBL InterferonSource)
  • Alkaline phosphatase–conjugated goat anti‐rabbit IgG
  • Alkaline buffer: 50 mM Na 2CO 3, 1 mM Mg 2Cl 2.6H 2O, pH 9.8 in water
  • p‐Nitrophenyl phosphate (Sigma)
  • 96‐well ELISA plates
  • 37°C incubator
  • Spectrofluorometer (405‐nm absorbance reading)

Alternate Protocol 2: Measurement of IFN by Bioassay

  • L929 murine fibroblast cells (ATCC #CCL‐1)
  • Serum‐free RPMI
  • Cocal virus of quantified titer (ATCC #VR‐435)
  • 5 mg/ml thiazolyl blue tetrazolium bromide (MTT)
  • 10% SDS, 0.1 % HCl in water
  • Neutralizing anti‐IFNγ Ab such as clone R4‐6A2 (BD Biosciences) or clone HB170 (Havell, )
  • Neutralizing anti‐IFNα/β Ab such as a combination of polyclonal anti‐IFNβ Ab and polyclonal anti‐IFNα Ab (PBL InterferonSource)
  • IFNγ standard (PBL InterferonSource)
  • 96‐well flat‐bottomed and U‐bottomed tissue culture plates
  • Spectrofluorometer (555 nm and 680 nm)
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Literature Cited

   Asselin‐Paturel, C., Brizard, G., Pin, J.J., Briere, F., and Trinchieri, G. 2003. Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody. J. Immunol. 171:6466‐6477.
   Asselin‐Paturel, C., Brizard, G., Chemin, K., Boonstra, A., O'Garra, A., Vicari, A., and Trinchieri, G. 2005. Type I interferon dependence of plasmacytoid dendritic cell activation and migration. J. Exp. Med. 201:1157‐1167.
   Boehm, U., Klamp, T., Groot, M., and Howard, J.C. 1997. Cellular responses to interferon‐gamma. Annu. Rev. Immunol. 15:749‐795.
   Chan, C.W., Crafton, E., Fan, H.N., Flook, J., Yoshimura, K., Skarica, M., Brockstedt, D., Dubensky, T.W., Stins, M.F., Lanier, L.L., Pardoll, D.M., and Housseau, F. 2006. Interferon‐producing killer dendritic cells provide a link between innate and adaptive immunity. Nat. Med. 12:207‐213.
   Diebold, S.S., Montoya, M., Unger, H., Alexopoulou, L., Roy, P., Haswell, L.E., Al‐Shamkhani, A., Flavell, R., Borrow, P., and Reis e Sousa, C. 2003. Viral infection switches non‐plasmacytoid dendritic cells into high interferon producers. Nature 424:324‐328.
   Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S., and Reis e Sousa, C. 2004. Innate antiviral responses by means of TLR7‐mediated recognition of single‐stranded RNA. Science 303:1529‐1531.
   Havell, E.A. 1986. Purification and further characterization of an anti‐murine interferon‐gamma monoclonal neutralizing antibody. J. Interferon. Res. 6:489‐497.
   Isaacs, A. and Lindenmann, J. 1957. Virus interference. I. The interferon. Proc. R. Soc. Lond. B Biol. Sci. 147:258‐267.
   Kawade, T. and Watanabe, Y. 1987. Characterization of rat monoclonal antibodies to mouse interferon‐α and ‐β. In Biology of the Interferon System pp. 197‐201. Proceedings of the Third International TNO Meeting on the Biology of the Interferon System. The Hague, The Netherlands.
   Seeds, R.E., Gordon, S., and Miller, J.L. 2009. Characterization of myeloid receptor expression and interferon alpha/beta production in murine plasmacytoid dendritic cells by flow cytomtery. J. Immunol. Methods 350:106‐117.
   Sjolin, H., Robbins, S.H., Bessou, G., Hidmark, A., Tomasello, E., Johansson, M., Hall, H., Charifi, F., Karlsson Hedestam, G.B., Biron, C.A., Karre, K., Hoglund, P., Vivier, E., and Dalod, M. 2006. DAP12 signaling regulates plasmacytoid dendritic cell homeostasis and down‐modulates their function during viral infection. J. Immunol. 177:2908‐2916.
   Symons, J.A., Alcami, A., and Smith, G.L. 1995. Vaccinia virus encodes a soluble type I interferon receptor of novel structure and broad species specificity. Cell 81:551‐560.
Key References
   Perler, L., Pfister, H., Schweizer, M., Peterhans, E., and Jungi, T.W. 1999. A bioassay for interferon type I based on inhibition of Sendai virus growth. J. Immunol. Methods 222:189‐196.
  Other helpful IFN bioassay protocols.
   Vogel, S.N., Friedman, R.M., and Hogan, M.M. 2001. Measurement of antiviral activity induced by interferons alpha, beta, and gamma. Curr. Protoc. Immunol. 37:6.9.1‐6.9.8.
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