The dynamics, nature, strength, and ultimately protective capabilities of an active immune response are determined by the extracellular constitution and concentration of various soluble factors. Generated effector cells secrete such mediators, including antibodies, chemo‐ and cytokines to achieve functionality. These secreted factors organize the individual immune cells into functional tissues, initiate, orchestrate, and regulate the immune response. Therefore, a single‐cell resolved analysis of protein secretion is a valuable tool for studying the heterogeneity and functionality of immune cells. This review aims to provide a comparative overview of various methods to characterize immune reactions by measuring single‐cell protein secretion. Spot‐based and cytometry‐based assays, such as ELISpot and flow cytometry, respectively, are well‐established methods applied in basic research and clinical settings. Emerging novel technologies, such as microfluidic platforms, offer new ways to measure and exploit protein secretion in immune reactions. Further technological advances will allow the deciphering of protein secretion in immunological responses with unprecedented detail, linking secretion to functionality. Here, we summarize the development and recent advances of tools that allow the analysis of protein secretion at the single‐cell level, and discuss and contrast their applications within immunology.

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