Optimization of whole blood antigen-specific cytokine assays for CD4(+) T cells

Cytometry. 2000 May 1;40(1):60-8. doi: 10.1002/(sici)1097-0320(20000501)40:1<60::aid-cyto8>3.0.co;2-j.


Background: The analysis of cytokine production is a valuable component of studies of immune response to stimulation such as pathogens, vaccines, and other immunological challenges. One highly sensitive method of cytokine evaluation involves three-color flow cytometric analysis of cytokine production in individual CD4(+) T cells.

Methods: We present four methods to enhance the acquisition and analysis of cells secreting the cytokines interferon gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and interleukin-4 (IL-4). Using cytomegalovirus (CMV) as the antigenic model, titration and kinetic experiments were carried out in whole blood from CMV-seropositive donors.

Results: CMV is most effective as a stimulating antigen when used at a dose of 5 microg/ml and for a period of at least 6 h, the first 2 h in the absence of 10 microg/ml Brefeldin A. This period of incubation can be made more convenient by the use of a "timed cooling" device, whereby the samples are automatically cooled and held at 4 degrees C at the end of incubation. Such timed cooling does not affect backgrounds or the proportion of responding cells. For certain samples, a high background can be reduced by adding fourth-color reagents. They identify and allow for elimination of monocytes and activated platelets, which contribute to false positive staining.

Conclusions: These optimizations make the assay both convenient for use in whole blood samples and highly reproducible (intra-assay variability is less than 10%; interassay variability is less than 25%).

MeSH terms

  • Antigens, Viral / immunology
  • Blood Donors
  • CD4 Lymphocyte Count / methods*
  • CD4-Positive T-Lymphocytes / chemistry
  • CD4-Positive T-Lymphocytes / cytology*
  • CD4-Positive T-Lymphocytes / virology
  • Cold Temperature
  • Cytomegalovirus / immunology
  • False Positive Reactions
  • Flow Cytometry / methods*
  • Flow Cytometry / standards
  • Fluorescent Antibody Technique
  • Humans
  • Interferon-gamma / analysis
  • Interleukin-2 / analysis
  • Interleukin-4 / analysis
  • Kinetics
  • Reproducibility of Results
  • Titrimetry
  • Tumor Necrosis Factor-alpha / analysis


  • Antigens, Viral
  • Interleukin-2
  • Tumor Necrosis Factor-alpha
  • Interleukin-4
  • Interferon-gamma