Cytokine secretion from T cells in response to antigen stimulation can be detected by measuring bulk cytokine production using an enzyme-linked immunosorbent assay or multiplexed flow cytometric assays, while cytokine assays at the single cell level can be performed by the enzyme-linked immunospot (ELISPOT) or cytokine flow cytometry (CFC) assays. In contrast to ELISPOT, CFC as a functional assay can be combined with other assays such as tetramer because both techniques can be applied to the same sample and analyzed by flow cytometry. In CFC, whole blood or peripheral blood mononuclear cells are incubated with antigen, usually in the presence of a secretion inhibitor such as brefeldin A or monensin, which allows intracellular accumulation of cytokines in activated cells. After fixation and permeabilization, the cells can be stained for intracellular cytokines and analyzed by flow cytometry.
BD Biosciences offers 3 different kits for fixation and permeabilization of cells for immunofluorescent staining of intracellular cytokines. I use the BD Cytofix/Cytoperm™ Plus Kit, which contains a fixation/permeabilization solution containing 4% paraformaldehyde, a permeabilization/washing solution and a protein transport inhibitor (GolgiPlug™ containing brefeldin A).
For CFC assays, cells are stimulated for 6 hours with 10 ug/ml peptide or 1 ug/ml Staphylococcus enterotoxin B, as a positive control, and then cells are kept overnight at 4ºC for convenience. GolgiPlug™ can be added in the beginning of stimulation or after 2 hours without any difference on cytokine production. Stimulated cells are harvested and stained for cell surface antigens such as CD3, CD4, and CD8. Cells are then fixed and permeabilized by thoroughly resuspending them in fixation/permeabilization solution for 20 minutes at 4ºC. Following 2 washing steps with the permeabilization/washing solution, cells can be stained for cytokine using an optimal concentration of a fluorochrome-conjugated anti-cytokine antibody or for an appropriate isotype as a negative control. It is essential to maintain using the permeabilization/washing solution during washing steps in order to keep the cells permeabilized. I usually perform IFN-gamma intracellular staining using R-Phycoerythrin-conjugated mouse anti-human IFN-gamma monoclonal antibody from BD Pharmingen (Catalog number: 554552). This monoclonal antibody works well with a low level of background.
In summary, cytokine flow cytometry assays are a vital tool for identification and enumeration of cytokine-secreting cells within mixed cell populations. Fixation, permeabilization and cytokine intracellular accumulation are all necessary steps for successful intracellular cytokine detection by flow cytometry, and I found all these are achievable using the BD Cytofix/Cytoperm™ Plus Kit.
Post-Doctoral Fellow
Paterson Institute for Cancer Research
University of Manchester