What is Intracellular Cytokine Staining?

A flow cytometry based assay that detects the production and accumulation of cytokines

Production of cytokines plays an important role in the immune response. Cytokines are involved in many different pathways including the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha. Cytokines are not preformed factors but are rapidly produced and secreted in response to cellular activation.

Intracellular cytokine staining  (ICS) is a very useful and widely used flow cytometry based assay which detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation.  ICS can be used in combination with other flow cytometry protocols for immunophenotyping using cell surface markers or with MHC multimers to detect an antigen specific response, making it an extremely flexible and versatile method.

The principle of intracellular cytokine staining is as follows:

  • Cells are activated using either a specific peptide or a non-specific activation cocktail
  • An inhibitor of protein transport (e.g. brefeldin A) is added to to retain the cytokines within the cell
  • After washing, antibodies to other cellular markers can be added to the cells
  • The cells are then fixed in paraformaldehyde and permeabilized
  • The anti-cytokine antibody is added and the cells can be analyzed by flow cytometer



Immune Monitoring

Both ICS and ELISpot can be used to monitor the same functional responses in human and animal studies. Whilst ICS generally requires more cells, the flexibility of the ICS assay allows a more in depth study of the cytokine producing cells. Another advantage over more traditional killing assays, is that ICS can be performed on cryo-preserved cells as well as fresh samples.

The major drawbacks of using ICS as a functional test is that the cells will activate in response to a wide variety of stimulatory signals and so it is difficult to unequivocally state that positive staining (especially low intensity staining) is truly antigen specific. Used in conjunction with other functional assays such as ELISpot or multimer staining, ICS can provide useful supporting evidence of an antigen specific response.

Epitope Discovery

The aim of epitope mapping projects is to identify and characterize novel epitopes from a protein that are recognized by the immune system. By identifying novel epitopes, strategies can be developed to produce immunotherapies or vaccines for a wide range of disease areas such as cancers or infectious diseases.

ICS can be used as part of an epitope discovery project, using pools of peptides. Due to the larger numbers of cells required for this assay, ELISpot is generally the preferred functional assay for screening large numbers of peptides. Once the number of prospective epitopes has been reduced, ICS is a good way to determine the response of the cells to a particular peptide, or small pool of peptides. As ICS can be easily combined with antibody and MHC multimer staining in flow cytometry is can provide useful phenotypic and functional data about antigen specific cells.