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 detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single-cell level. It detects the production and accumulation of cytokines within the endoplasmic reticulum after cell stimulation, allowing direct TH1 versus TH2 determination. It can also 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. This capability, combined with the high throughput nature of the instrumentation, gives intracellular cytokine staining an enormous advantage over existing single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The principle steps of intracellular cytokine staining is as follows:
1. Cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail;
2. An inhibitor of protein transport (e.g. Brefeldin A) is added to retain the cytokines within the cell;
3. Next, EDTA is added to remove adherent cells from the activation vessel;
4. After washing, antibodies to cell surface markers can be added to the cells;
5. The cells are then fixed in paraformaldehyde and permeabilized;
6. The anti-cytokine antibody is added and the cells can be analyzed by flow cytometer.
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