Figure 12 Cytokine profiles of human peripheral T cells. Human T cells were either resting (A) or stimulated with phorbol ester and ionomycin - a powerful activating combination (B). After 6 h, cells were stained for the presence of intracellular cytokines (i.e. the cytokines that are just synthesized and are about to be secreted for effector functions). Essentially no resting T cells are synthesizing cytokines; stimulated T cells will make either interleukin 4 (IL-4) or interferon y (IFNy) but very rarely both. Note that a bulk measurement of cytokine production would reveal that both cytokines are made in the population, but would not demonstrate the exclusivity of production on a cell-by-cell basis.

production are typically performed after at least 24 h of culture and provide no information about the distribution of cytokine expression among the cells (e.g. are all cells making the same amount of cytokine, or are only a small fraction of the cells each making a lot?). In the example of Figure 12, it would be impossible to deduce from a bulk assay that some cells make only IL-4, while others make only IFNy.

Again, the real utility of the FACS for making functional assays comes with the combination of these assays with each other or with immunopheno-typing. Thus, not only is it possible to measure how many cells make a particular cytokine, but also what the phenotype of those cells is, and, for instance, what their proliferative capacity is.

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