Measurement of Human and Murine Interleukin 2 and Interleukin 4

Laurie S. Davis1, Peter E. Lipsky1, Kim Bottomly2

1 University of Texas Southwestern Medical Center, Dallas, Texas, 2 Yale University School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.3
DOI:  10.1002/0471142735.im0603s37
Online Posting Date:  May, 2001
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Abstract

This unit describes protocols employing cell lines or bioassays that can be used for the quantitation of murine total T cell growth factor (TCGF) activity, interleukin 2 (IL‐2), and interleukin 4 (IL‐4), and of human IL‐2 and IL‐4. The ability to distinguish between different growth factors is crucial to understanding the regulation of the immune response. The describes the use of the CTLL‐2 line to detect murine IL‐2 and IL‐4 in supernatants. One alternate protocol describes the detection of IL‐2 in samples of human serum or supernatants using CTLL‐2 cells, while other alternate procedures describe the detection and quantitation of murine IL‐4 using a mutagenized subline of CTLL‐2, CT.4S, and the detection of human IL‐4 using a derivative of the CT.4S mouse cell line, CT.h4S. Support protocols are provided for the quantitation of CTLL‐2, CT4.S, or CT.h4S proliferation using a standard [3H]thymidine incorporation method or by using the 3‐(4,5‐dimenthylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay (). Support protocols also describe the calculation of cytokine units from samples based on DNA synthesis data and procedures for the maintenance of the CTLL‐2, CT.4S, and CT.h4S cell lines.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Murine IL‐2 and IL‐4 Using CTLL‐2 Cells
  • Alternate Protocol 1: Detection of Human IL‐2 Using CTLL‐2 Cells
  • Alternate Protocol 2: Detection of Murine IL‐4 Using CT.4S Cells
  • Alternate Protocol 3: Detection of Human IL‐4 Using CT.h4S Cells
  • Support Protocol 1: Quantitation of DNA Synthesis Using the MTT Colorimetric Assay
  • Support Protocol 2: Calculation of Interleukin or T Cell Growth Factor (TCGF) Units
  • Support Protocol 3: Maintenance of Murine CTLL‐2 Cells
  • Support Protocol 4: Maintenance of Murine CT.4S Cells
  • Support Protocol 5: Maintenance of Murine CTh.4S Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of Murine IL‐2 and IL‐4 Using CTLL‐2 Cells

  Materials
  • Recombinant murine IL‐2 (Amgen) and IL‐4 (Genzyme)
  • Complete RPMI‐10 medium ( appendix 2A)
  • CTLL‐2 T cells (ATCC)
  • Lymphocyte separation medium (LSM; Organon Teknika Cappel)
  • [3H]thymidine ( appendix 3D)
  • IL‐2 blocking antibody (rat anti‐mouse IL‐2, S4B6; ATCC)
  • IL‐4 blocking antibody (rat anti‐mouse IL‐4, 11B11; ATCC)
  • 96‐well flat‐bottom microtiter plates (Costar)
  • Sorvall RT6000B centrifuge and 50‐ml centrifuge tube
  • Additional reagents and equipment for maintenance of murine CTLL‐2 cells (see protocol 7), trypan blue exclusion ( appendix 3B), enrichment of viable cells (unit 3.1), labeling and harvesting cells and determining [3H]thymidine incorporation ( appendix 3D), MTT colorimetric quantitation of DNA synthesis (see protocol 5), and calculation of cytokine units in samples (see protocol 6)

Alternate Protocol 1: Detection of Human IL‐2 Using CTLL‐2 Cells

  • Recombinant human IL‐2 (Amgen)
  • PBS ( appendix 2A), Hanks balanced salt solution (HBSS; appendix 2A), or saline solution

Alternate Protocol 2: Detection of Murine IL‐4 Using CT.4S Cells

  • Recombinant human IL‐4 (R & D Systems)
  • CT.h4S cells
  • PBS ( appendix 2A)
  • Cell scraper

Alternate Protocol 3: Detection of Human IL‐4 Using CT.h4S Cells

  • MTT (Sigma)
  • PBS ( appendix 2A)
  • 0.04 N HCl in isopropanol
  • Filter unit (Millipore)
  • Microtiter plate reader with 570‐nm filter (and, optionally, 630‐nm filter)

Support Protocol 1: Quantitation of DNA Synthesis Using the MTT Colorimetric Assay

  • 75‐cm2 tissue culture flasks (Corning)
  • Recombinant human IL‐2 (Amgen)

Support Protocol 2: Calculation of Interleukin or T Cell Growth Factor (TCGF) Units

  • 1 mM sodium pyruvate (GIBCO/BRL)
  • Recombinant human IL‐4 (R&D Systems)
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Figures

Videos

Literature Cited

Literature Cited
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   Gearing, A.J.H. and Bird, C.R. 1987. Production and assay of interleukin 2. In Lymphokines and Interferons: A Practical Approach (M.J. Clemens, A.G. Morris, and A.J.H. Gearing, eds.) pp. 291‐301. IRL Press, Oxford.
   Gillis, S., Ferm, M.M., Ou, W., and Smith, K.A. 1978. T cell growth factor:Parameters of production and a quantitative microassay for activity. J. Immunol. 120:2027‐2032.
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   Kinzer, C.A., Keegan, A.D., Beckmann, M.P., Widmer, M., Idelson, G.H., Ben‐Sasson, S.Z., and Paul, W.E. 1994. CT.h4S cells, a murine cell line expressing human IL‐4 receptors and capable of assaying human IL‐4. FASEB J. 8:A506.
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   Xu, H., Rizzo, L.V., and Caspi, R.R. 1995. IL‐12 induces growth of the IL‐4‐dependent CT4S line and has a synergistic effect on IL‐4‐induced CT4S proliferation. J. Immunol. Methods 181:245‐251.
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