UNIT 6.24 Detection of Intracellular Cytokines by Flow Cytometry

  1. Barbara Foster,
  2. Calman Prussin

Published Online: 1 NOV 2002

DOI: 10.1002/0471142735.im0624s50

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Foster, B. and Prussin, C. 2002. Detection of Intracellular Cytokines by Flow Cytometry. Current Protocols in Immunology. 6:6.24.

Author Information

  1. National Institute of Allergy and Infectious Diseases/NIH, Bethesda, Maryland

Publication History

  1. Published Online: 1 NOV 2002
  2. Published Print: AUG 2002

This is not the most recent version of the article. View current version (3 AUG 2015)


Intracellular cytokine detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single-cell level. Multiparameter flow cytometry permits simultaneous detection of two or more cytokines within a single cell, allowing direct TH1 versus TH2 determination. This capability, combined with the high throughput inherent in the instrumentation, gives intracellular cytokine staining an enormous advantage over existing single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes the techniques required to perform intracellular staining of cells that have already been stimulated in vitro and fixed. The methods necessary for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell-surface staining are also described. Because of the greater level of nonspecific binding inherent in fixed, permeabilized cells, greater care must be taken in designing specificity controls. A more rigorous method to verify specificity of staining is also described.