Intracellular cytokine detection by fluorescence-activated flow cytometry: Basic principles and recent advances

Intracellular cytokine staining is a flow cytometric technique consisting of culturing stimulated cytokine-producing cells in the presence of a protein secretion inhibitor, followed by fixation, permeabilization and staining of intracellular cytokines and cell markers (surface or cytoplasmic) with fluorescent antibodies. Up to 18 different colors can be detected by modern flow cytometers, making it the only immunological technique allowing simultaneous determination of antigen-specific T cell function and phenotype. In addition, cell proliferation and viability can be also measured. For this reason, it is probably the most popular method to measure antigenicity during vaccine trials and in the study of infectious diseases, along with ELISPOT. In this review, we will summarize its features, provide the protocol used by most laboratories and review its most recent applications.