FluoroSpot assay

The dual-color FluoroSpot assay is a modification of the ELISPOT assay and developed for the simultaneous detection of two cytokines released by a single T cell. The assay is based on the use of antibodies labeled with different fluorophores enabling the detection of T cells producing two pre-selected cytokines. The Fluorospot assay is particularly useful for the analysis of T cell subpopulations with distinct cytokine profiles.
U-CyTech biosciences offers a wide range of human and monkey cytokine FluoroSpot kits including kits for the combinatorial detection of IFN-γ (interferon-gamma) and a secondary cytokine (e.g. IL-2, IL-10, IL-13). Our monkey FluoroSpot kits cross react with serveral macaque and baboon species. These kits are supplied with PVDF membrane-bottomed (PVDF) plates, which are specifically designed for the FluoroSpot assay. 


  • The Fluorospot assay is an effective tool to enumerate antigen-specific T cells with simultaneous detection of two different cytokines in the circulation of (non) immunized humans and animals with a high sensitivity.
  • Since the Fluorospot assay is able to detect 2 cytokines simultaneously in one assay, the necessary amount of PBMCs is lower.  Therefore, the Fluorospot is an ideal tool for clinical trials, in which a limited amount of PBMCs are available.


Brief description of the FluoroSpot assay

Cells are incubated in the wells of the FluoroSpot plate precoated with two high-affinity antibodies specific for different cytokines. After binding of the released cytokines, cells are washed away. Areas in which the cytokines have been captured by the two coating antibodies are detected by incubation with a mixture of two different anti-cytokine detector antibodies followed by an incubation step with two fluorescent-labeled conjugates. The generation of spots that emit light at different wavelengths enables the precise localization of single and double cytokine-producing cells. Green and red fluorescent spots represent cells that produce different cytokines whereas yellow spots originate from cells that release both cytokines.


U-CyTech FluoroSpot products

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