The FluoroSpot assay was developed as an adaptation of traditional enzymatic ELISPOT detection systems using colorimetric substrates. Utilizing fluorochrome-conjugated detection antibodies, the FluoroSpot is capable of detecting secreted cytokine or antibody (for B cell FluoroSpot) with the same high level of sensitivity as in ELISPOT.


The most significant advantage of FluoroSpot assays is their ability to detect multiple different cytokines (or secreted Immunoglobulin classes and sub-classes in B cell FluoroSpot) simultaneously in the same assay well. With enzymatic/colorimetric substrate development systems as used in ELISPOT, the limitations of the method restrict the number of analytes to a maximum of two. Whereas Double-Color Enzymatic ELISPOT assays have been shown to be highly accurate and comparable in sensitivity to single-analyte detection done in parallel, the use of 2, 3 or even 4 color FluoroSpot has become more feasible and offers many advantages for researchers, while retaining the accuracy and sensitivity of classic ELISPOT assay methodologies.

The multi-parametric potential of FluoroSpot opens up the possibility for researchers to examine, with the same level of ease and sensitivity as in ELISPOT, additional functional and qualitative parameters of T cell-mediated immune responses. FluoroSpot assays, measuring the different cytokines and effector molecules secreted by activated T cells, can provide invaluable information on the effector lineage(s) of T cell immunity. For example, an area of increasing interest recently has been in the study of polyfunctional T cells – cells which can be shown to secrete multiple different cytokines.

Using an IFN-γ / IL-2 FluoroSpot assay system, T cells secreting IFN-γ, IL-2, as well as cells that secrete both cytokines, can be individually detected and visualized simultaneously in the same assay well. In this case, IFN-γ secreting cells will produce green spots, and IL-2 producing cells will produce red spots. Polyfunctional cells secreting both analytes will appear yellow, as the two fluorescent spectra from IFN-γ and IL-2 are overlapping. In a FluoroSpot assay, the frequencies of antigen specific T cells responding to stimulation reflect the magnitude of T cell immunity, as in classic ELISPOT assays. The spot sizes and intensities measured by the FluoroSpot Analyzer record the amount of cytokine produced by individual secreting cells.

In FluoroSpot assays, given the means of fluorescence detection, as measured by the intensity of the signal of a specific wavelength, these amounts can be quantified, as opposed to ELISPOT. ImmunoSpot Fluoro-X FluoroSpot analysis software can quantify the spot sizes and intensities to give researchers a means to measure and compare the relative robustness of individual T cells. Spot size also indicates the functionality of the responding T cell. Spot sizes and intensities can be increased in polyfunctional T cells as well as in recently activated memory effector T cells. Likewise, spot sizes and intensities can be decreased in states of anergy or immune suppression.

Polyfunctional CD8+ T cells secreting 2 or more cytokines seem to confer greater levels of protective immunity, whereas CD8+ T cells secreting a single cytokine are not necessarily as effective in clearing virus, nor at providing protection against re-challenge with virus. High numbers of IFN-g/IL-2 secreting polyfunctional T cells correlate with protection again viral infection, whereas cells secreting either cytokine alone did not correlate with protection. The ability to control progression of viral infection has thus been strongly correlated with the quality of the immune response, i.e. the functionality of the CD8+ T cell response to the virus. High ratios of IFN-γ, IL-2, TNF-α, MIP-1β, co-expressing T cells to single cytokine producers strongly correlate with viral clearance and/or non-progression of the disease, and thus with functional T-cell responses.

These complex inter-related co-expression patterns are a subject of intense ongoing study, and FluoroSpot is ideally suited for the investigation of such polyfunctional T cells. FluoroSpot assays can visualize and enumerate the relative numbers of polyfunctional T cells and single-cytokine producers in single assay well. Using fluorescent labels with discrete, non-overlapping wavelengths, individual cytokines can be easily differentiated, as well as co-secreted cytokines.

FluoroSpot assays are an effective method for visualizing and enumerating T helper cells of different types within a PBMC or isolated or enriched CD4+ T cell population. Dual-color FluoroSpot assays can also provide the researcher a simple and fast means of distinguishing between Th1 and Th2 type cells in the same assay well. For instance, an IFN-γ / IL-4 FluoroSpot can distinguish Th1 and Th2 cells. Likewise, Th1 vs. Th17, or Th1 vs. Treg (T reg) cell populations can be visualized, and the frequencies of each type determined.

B cell FluoroSpot

Another useful application for FluoroSpot assays is in determining populations of Antibody Secreting Cells secreting different Immunoglobulin (Ig) classes and IgG sub-classes within the same assay well. Here, the frequencies of different Ig classes and/or sub-classes can be investigated, giving the researcher a powerful investigative tool.