Intracellular Cytokines

George F. Babcock1

1 University of Cincinnati College of Medicine and Shriners Burns Institute, Cincinnati, Ohio
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.9
DOI:  10.1002/0471142956.cy0909s28
Online Posting Date:  May, 2004
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The principal technique described in this unit was developed for detecting cytokines produced by T cells, specifically interferon gamma (IFN‐γ) and interleukin 4 (IL‐4). In addition to providing information about which cytokines are being produced, it also helps to phenotypically identify the specific cells producing them. For example, CD4+ or CD8+ cells can be subdivided into TH1/TH2 helper cells or TC1/TC2 suppressor cells, respectively. This procedure can be useful in examining the response of cells to a variety of agonists, the immune function in various disease states, and the level of lymphocyte activation or suppression. In addition, it can be used to detect a variety of cytokines in many cell types, although the methodology must be carefully evaluated for each cytokine or cell type of interest. Additional methods are included for the stimulation of T cells with allogeneic cells and for the performance of controls for labeling specificity.

Keywords: T cells; cytokines; immune function; lymphocyte activation; interferon gamma; interleukin 4

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Table of Contents

  • Basic Protocol 1: Measurement of Intracellular Interferon Gamma and Interleukin 4 in T Lymphocytes
  • Support Protocol 1: Stimulation of T Cells with Alloantigen
  • Support Protocol 2: Testing Specificity of Cytokine Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Measurement of Intracellular Interferon Gamma and Interleukin 4 in T Lymphocytes

  • Rat, mouse, or human whole blood or rat or mouse spleen cells (Kruisbeek, )
  • Heparin‐containing Vacutainer tubes (unit 9.7) or syringe containing sodium heparin at 20 U/ml blood
  • Cell culture medium (see recipe)
  • 10 µg/ml PMA (see recipe)
  • 250 µg/ml A23187 (see recipe)
  • 1 mM monensin (see recipe) or 100 µg/ml brefeldin A (see recipe)
  • Wash buffer (see recipe), ice cold
  • 20 µg/ml murine gamma globulin (see recipe) ice cold
  • Fluorochrome‐labeled antibodies (see recipe; anti‐cytokine, anti–cellular antigen, and/or isotypic controls), or kit for intracellular cytokine staining (see recipe)
  • 1% and 4% buffered fixative (see recipe)
  • Permeabilizing solution (one of the following): 0.1% saponin (see recipe), FACS permeabilizing solution (Becton Dickinson Immunocytometry), Fix and Perm (Caltag Labs), Cytofix/Cytoperm (PharMingen), Cell Permeabilization Kit (Harlan Bioproducts for Science), Cytoperm (Serotech), or Permeafix (Ortho)
  • 12 × 75–mm polystyrene or polypropylene snap‐capped tubes
  • Centrifuge (Beckman GS‐6R with swinging bucket rotor, or equivalent)
  • CO 2 incubator at 37°C
  • Additional reagents and equipment for stimulation with allogeneic cells (see protocol 2) and for flow cytometry (unit 6.2)

Support Protocol 1: Stimulation of T Cells with Alloantigen

  • Allogeneic lymphocytes
  • 500 µg/ml mitomycin C (optional; prepare in DPBS and store up to 2 weeks in the dark at 4°C)
  • Dulbecco's phosphate‐buffered saline (DPBS, e.g. Life Technologies; prepare according to manufacturer's instructions), ice cold
  • 5% CO 2/95% air incubator at 37°C

Support Protocol 2: Testing Specificity of Cytokine Staining

  • Purified cytokine
  • Unconjugated anti‐cytokine antibody
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Literature Cited

   Czerkinsky, C., Nilsson, L., Nygren, H., Ouchterlony, Ö., and Tarkowski, A. 1983. A solid phase enzyme‐linked immunospot (ELISPOT) assay for enumeration for specific antibody‐secreting cells. J. Immunol. Methods 65:109‐121.
   Elston, L., Nutman, T., Metcalfe, D., and Prussin, C. 1995. Flow cytometric analysis for cytokine production identifies T helper 1 and T helper 2 and T helper 0 cells within the human CD4+ CD27− lymphocyte population. J. Immunol. 154:4294‐4301.
   Frede, S.E., Valente, J., Alexander, J.W., and Babcock, G.F. 1997. The relationship of blood transfusion and immunosuppression with the Th1/Th2 paradigm. Transplant. Proc. 29:1153‐1154.
   Kruisbeek, A.M. 1993. Isolation of mouse mononuclear cells. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and, W. Strober, eds.) pp. 3.1.2‐3.1.5. John Wiley & Sons, New York.
   Rodeberg, D.E., Morris R.E., and Babcock, G.F. 1997. Azurophilic granules of human neutrophils contain CD14. Infect. Immun. 65:4747‐4753.
   Schuerwegh, A., DeClerck, L., Bridts, C., and Stevens, W. 2003. Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques. Cytometry 55B:52‐58.
Key References
   Levy, A.E., Alexander, J.W., and Babcock, G.F. 1997. A strategy for generating consistent long term donor specific tolerance to solid organ allografts. Transplant. Immunol. 5:83‐88.
  Application of this protocol for detection of TH1 and TH2 cells.
   Prussin, C. and Metcalfe, D. 1995. Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti‐cytokine antibodies. J. Immunol. Methods 188:117‐128.
  Description of basic method and various fluorochromes that can be used.
   Prussin, C. 1997. Cytokine flow cytometry: Understanding cytokine biology at the single‐cell level. J. Clin. Immunol. 17:195‐204.
  An excellent review and troubleshooting guide.
   Sander, B., Andersson, J., and Andersson, U. 1991. Assessment of cytokines by immunofluorescence and the paraformaldehyde‐saponin procedure. Immunol. Rev. 119:65‐93.
  First description of the use of cytometry to measure intracellular cytokines.
   Vikingson, A., Pederson, K., and Muller, D. 1994. Enumeration of IFN‐γ producing lymphocytes by flow cytometry and correlation with quantitative measurement of IFN‐γ. J. Immunol. Methods 173:219‐228.
  Explanation of protocol and comparison of intracellular staining with other methods.
Internet Resources
  Web site of Becton Dickinson Biosciences
  Web site of BioLegend.
  Web site of Biozol (Eching, Germany).
  Web site of eBioscience.
  Web site of R & D Systems.
  Web site of Molecular Probes.
  Web site of Prozyme.
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