Figure 1.

Schematic representation of the different technologies enabling analysis of protein secretion at the single‐cell level, ranging from spot‐, cytometry‐, well‐, chamber‐ and droplet‐based assays, of which the last three are summarized as microfluidic methods in this review. Common features of all described technologies are spatial separation of the individual cells, and their reliance on labeled reporters, especially antibodies and variants thereof. However, all the described technologies differ in several respects, e.g., in the read‐out obtained, ease of application, multiplexing potential, cellular throughput, temporal resolution, and other characteristics that need consideration when a specific technique is used to analyze a sample. Therefore, all technologies have different applications and limitations (see also Table 1). The spot‐based assays ELISpot and FluoroSpot, based on the seeding of a heterogeneous population of cells on a protein‐binding membrane, allow for the rapid and simple determination of the frequency of secreting cells based on an antibody‐based immunoassay. The frequency of cells stained positively with a detection reagent can be assessed using cytometry‐based methods such as flow cytometry and mass cytometry. With this technology, the heterogeneous population of cells is measured temporally and spatially separated from each other, and positive events are gated and counted as a frequency of total input cells. The three microfluidic methods shown on the right are all based on the same concept of individually trapping and analyzing cells in small volumes. Here, the secreted molecule rapidly reaches a detectable concentration due to the small volume, allowing more accurate and rapid quantification of protein secretion. The microfluidic methods can be further divided based on the enclosing structure used. Well‐based assays encapsulate cells in small circular or square wells, whereas chamber‐based use elongated chambers that fit a higher volume and allow spatially separating the detection of various secreted proteins. Lastly, droplet‐based assays use two immiscible fluids to generate an emulsion to encapsulate individual cells in small volumes, and enable a surface‐free, volume‐based analysis of protein secretion in high‐throughput and with high precision.