Measurement of Interleukin 6

Richard P. Nordan1, C.D. Richards2, Jack Gauldie2

1 National Cancer Institute, Bethesda, Maryland, 2 McMaster University, Hamilton, Ontario, Canada
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.6
DOI:  10.1002/0471142735.im0606s17
Online Posting Date:  May, 2001
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Abstract

Interleukin 6 (IL‐6) is a pluripotent cytokine with multiple effects on many different cell types. It is produced by a variety of cells in response to immunological and other stimuli. This unit describes a simple and sensitive assay for human, rat, rabbit, pig, and mouse IL‐6, based on IL‐6‐dependent proliferation of a murine B cell hybridoma cell line, B9. Support protocols discuss maintenance of B9 cells, preparation of IL‐6 standards, and production of IL‐6‐containing supernatant. In addition, IL‐6 ELISA kits for the measurement of human or mouse IL‐6 are available from a number of companies. These products are reliable, highly sensitive, and specific, and thus should be considered as an excellent (although more expensive) alternative, keeping in mind that bioactivity is not assessed with this approach

     
 
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Table of Contents

  • Basic Protocol 1: B9/Hybridoma Growth Factor Assay
  • Support Protocol 1: Preparation of IL‐6‐Containing Supernatant
  • Support Protocol 2: B9 Cell Maintenance
  • Support Protocol 3: Preparation of IL‐6 Standards
  • Commentary
     
 
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Materials

Basic Protocol 1: B9/Hybridoma Growth Factor Assay

  Materials
  • B9 cells in log phase of growth (see protocol 3)
  • Complete RPMI‐10 medium ( appendix 2A)
  • IL‐6 standard (see protocol 4)
  • [3H]thymidine ( appendix 3D) or MTT (unit 6.3)
  • 96‐well flat‐bottomed microtiter plates
  • 15‐ml screw‐cap tubes, sterile
  • 12‐channel pipettor (50‐ to 250‐µl adjustable volume; ICN Biomedicals)
  • V‐bottom reservoir trays (Robbins Scientific), sterile
  • Additional reagents and equipment for counting and harvesting cells and determination of DNA synthesis by [3H]thymidine incorporation ( appendix 3D) or MTT colorimetric assay (unit 6.3)

Support Protocol 1: Preparation of IL‐6‐Containing Supernatant

  • Murine macrophage cell line P388D1 (ATCC #TIB 63) or human fibroblasts MRC‐5 (ATCC #CCL‐171)
  • RPMI‐5 (RPMI 1640 medium with 5% FBS heat‐inactivated 1 hr at 56°C) and serum‐free RPMI 1640, or DMEM‐10 and DMEM‐5 (DMEM with 10% and 5% heat‐inactivated FBS)

Support Protocol 2: B9 Cell Maintenance

  • Human or mouse IL‐6 stock: IL‐6‐containing supernatant from cell culture (see protocol 2) or recombinant IL‐6 (R & D Systems)
  • B9 cells (for source, see above paragraph)
  • 10% dimethyl sulfoxide (DMSO)/90% heat‐inactivated FBS
  • 25‐cm2 tissue culture flask
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Figures

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Literature Cited

Literature Cited
   Aarden, L.A., De Groot, E.R., Schaap, O.L., and Lansdorp, P.M. 1987. Production of hybridoma growth factor by human monocytes. Eur. J. Immunol. 17:1411‐1416.
   Garman, R.D., Jacobs, K.A., Clark, S.C., and Raulet, D.H. 1987. B‐cell‐stimulatory factor 2 (B2‐interferon) functions as a second signal for interleukin 2 production by mature murine T‐cells. Proc. Natl. Acad. Sci. U.S.A. 84:7629‐7633.
   Gauldie, J., Richards, C., Harnish, D., Lansdorp, P., and Bauman, H. 1987. IFN‐β2/BSF‐2 shares identity with monocyte‐derived hepatocyte stimulating factor (HSF) and regulates the major acute phase protein response in liver cells. Proc. Natl. Acad. Sci. U.S.A. 84:7251‐7255.
   Hirano, T., Yasukawa, H., Harada, T., Taga, T., Watanabe, Y., Matsuda, T., Kashiwamura, S., Nakajima, K., Koyama, K., Iwamatsu, A., Sakiyama, F., Matsui, H., Takahara, Y., Yaniguchi, T., and Kishimoto, T. 1986. Complementary DNA for a novel human interleukin (BSF‐2) that induces B lymphocytes to produce immunoglobulin. Nature 324:73‐76.
   Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55‐63.
   Nordan, R.P. and Potter, M. 1986. A macrophage‐derived factor required by plasmacytomas for survival and proliferation in vitro. Science 233:566‐569.
   Van Snick, J., Cayphas, S., Vink, A., Uyttenhove, C., Coulie, P., and Simpson, R. 1986. Purification and NH2‐terminal amino acid sequence of a new T cell‐derived lymphokine with growth factor activity for B cell hybridomas. Proc. Natl. Acad. Sci. U.S.A. 83:9679‐9683.
   Zilberstein, A., Ruggieri, R., Korn, J.H., and Revel, M. 1986. Structure and expression of cDNA and genes for human‐interferon‐β2, a distinct species inducible by growth‐stimulatory cytokines. EMBO J. 5:2529‐2537.
Key References
   Aarden, et al., 1987. See above.
  The original article from which the was developed.
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