This assay is used to look for the responsiveness of T cells to a specific antigenic stimulation. Stimulation can be performed with mononuclear cells isolated from PBMC or whole blood. The assay is based on the principle by treating cells with an inhibitor of the secretory pathway to prevent secretion of the cytokine (e.g brefeldin A or monensin) the cytokines accumulate inside the cytoplasm and can be detected by staining the fixed and permeabilized cells. By combining the intracellular cytokine stain with staining for phenotypic markers (as well as tetramers) it is possible to determine the type of cells that produce the cytokine as well as the quantity of cytokine produced per cell. This assay requires larger sample volumes than the ELISPOT but is the only assay that determines simultaneously the type of cytokine produced by a single cell and the phenotype of such a cell.