Flow cytometric visualisation of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood

Cytometry. 2000 Jan 1;39(1):72-8. doi: 10.1002/(sici)1097-0320(20000101)39:1<72::aid-cyto10>3.0.co;2-r.


Background: Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring.

Methods: With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population.

Results: Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation.

Conclusions: This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brefeldin A / pharmacology
  • CD3 Complex / analysis
  • CD56 Antigen / analysis
  • Cytokines / analysis*
  • Flow Cytometry / methods*
  • Humans
  • K562 Cells
  • Killer Cells, Natural / drug effects
  • Killer Cells, Natural / metabolism*
  • Monensin / pharmacology
  • Protein Synthesis Inhibitors / pharmacology
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / metabolism*


  • CD3 Complex
  • CD56 Antigen
  • Cytokines
  • Protein Synthesis Inhibitors
  • Brefeldin A
  • Monensin