FluoroSpot

FluoroSpot is a plate-based immunoassay derived from the ELISpot technique that uses fluorescent detection to quantify cytokine-secreting cells at the single-cell level. In a typical FluoroSpot assay, immune cells are placed in wells coated with capture antibodies against specific cytokines. After stimulation, secreted cytokines are captured on the membrane, and fluorescently labeled detection antibodies bind them. Spots corresponding to individual cytokine-secreting cells are then detected and counted using a fluorescence reader or imaging system.

A key feature of FluoroSpot is multiplexing. Different cytokines are labeled with spectrally distinct fluorophores, allowing simultaneous readout of multiple analytes in the same well. This enables analysis of polyfunctional T-cell responses and identification of cells that secrete combinations of cytokines. FluoroSpot can also be adapted for detecting antibody-secreting cells by using anti-immunoglobulin detection reagents.

Applications of FluoroSpot include vaccine evaluation, infectious disease research, cancer immunology, autoimmune studies, and transplant immunology. It is used to profile cellular immune responses and assess the functional quality of those responses, including the frequency and co-expression patterns of cytokines.

Advantages and limitations are noted in practice. Benefits include multiplex capability and rich single-cell information, enabling detailed functional profiling. Limitations involve the need for fluorescence instrumentation and careful spectral planning to manage overlap between fluorophores, as well as more complex optimization, poorer signal-to-noise in some contexts, and higher cost compared with single-analyte formats.

History-wise, FluoroSpot was developed as a fluorescence-based alternative to traditional ELISpot to enable simultaneous detection of multiple cytokines, with commercial kits and protocols contributing to its broad use in immunology research.