FluoroSpot is the multiplex version of ELISpot, enabling the simultaneous detection of multiple antigen‑specific immune responses at the single‑cell level. Like ELISpot, FluoroSpot identifies individual cells actively secreting cytokines, antibodies, or other immune mediators—but uses fluorescent detection to distinguish cells secreting one or several analytes in the same well.
Mabtech FluoroSpot kits are widely used in research and clinical development to study polyfunctional T‑ and B‑cell responses. Optimized reagents, low‑fluorescence plates, and validated antibody combinations ensure reliable multiplex performance, even when working with limited or precious samples.
Key Advantages
- Multiplex detection – Simultaneous analysis of up to four analytes in the same well
- Exceptional sensitivity – Detects rare antigen‑specific T‑ or B‑cell responses
- Single‑cell resolution – Direct identification of mono‑ and polyfunctional immune cells
- Functional profiling – Distinguish cells secreting one or multiple cytokines
- Reproducible performance – Optimized components ensure consistent results across experiments
So how does FluoroSpot work?
FluoroSpot follows the same basic principle as ELISpot but uses fluorescent detection instead of enzymatic visualization. Immune cells are stimulated in wells pre‑coated with capture antibodies specific to the analytes of interest. Responding cells secrete cytokines or antibodies, which are immediately captured on the membrane. After incubation, the cells are washed away and fluorophore‑labeled detection antibodies are added.
Spots are visualized using a fluorescence reader. Overlapping fluorescent signals identify cells secreting multiple analytes, allowing direct analysis of polyfunctional immune responses at the single‑cell level.
Want to learn more about the FluoroSpot assay principle?
Applications
T‑Cell FluoroSpot
FluoroSpot is used to investigate antigen‑specific T‑cell responses and functional diversity across many research areas, including:
- Infectious disease research (e.g. viral, bacterial, and parasitic immunity)
- Cancer and immuno‑oncology
- Allergy and autoimmune disease studies
- Vaccine development and evaluation
By enabling simultaneous detection of cytokines such as IFN‑γ, IL‑2, TNF‑α, and Granzyme B, FluoroSpot provides deeper insight into T‑cell polyfunctionality than single‑analyte assays.
Immunotherapy
FluoroSpot is a valuable functional assay in the development of CAR‑T cells, TIL therapies, and gene‑modified immune cells. Multiplex detection enables sensitive assessment of cytokine secretion profiles and supports functional characterization aligned with translational and regulatory expectations.
Vaccines
In vaccine research and development, FluoroSpot is commonly applied to evaluate antigen‑specific T‑cell responses and polyfunctionality. The assay allows researchers to investigate immune memory, identify relevant vaccine epitopes, assess adjuvant effects, and monitor the quality, magnitude, and durability of cellular immune responses following immunization.
B‑Cell FluoroSpot
B‑cell FluoroSpot enables the analysis of humoral immune responses at the single‑cell level by detecting antibodies at the moment of secretion, rather than in serum. The assay allows simultaneous assessment of multiple immunoglobulin isotypes and subclasses, providing detailed insight into B‑cell functionality.
This approach allows researchers to:
- Quantify antibody‑secreting cells (ASCs)
- Evaluate memory B‑cell responses after vaccination or infection
- Characterize immunoglobulin isotypes and subclasses
- Detect antigen‑specific B‑cell activity using native antigens
Evaluate cross‑reactive B cells
B‑cell FluoroSpot can be used to address key research questions in infection and vaccine research by enabling analysis of multiple antigens in the same assay. Using reverse B‑cell FluoroSpot, different antigens can be applied to identify both single‑reactive and cross‑reactive B cells at the single‑cell level.
This added layer of resolution supports deeper characterization of B‑cell specificity and cross‑reactivity, helping to guide antigen selection, vaccine design, and immune response evaluation.
Memory responses can be assessed following in‑vitro stimulation of PBMCs, providing valuable insight into long‑term humoral immunity.
FluoroSpot kit formats
FluoroSpot Flex | FluoroSpot Plus | FluoroSpot Path |
|---|---|---|
| Build your own kit | Reproducible | Antigen-specific |
Build your own FluoroSpot kit with thousands of combinations to choose from. Choose your analytes, and the appropriate plates and stimuli are included. Total assay time: Kit size options: | Our most popular FluoroSpot kits with validated analyte combinations and everything you need for the assay including a positive control (for human and NHP kits). Total assay time: Kit size options: | A complete FluoroSpot kit to answer your specific scientific question with validated analyte combinations, an included PepPool and positive control. Just add cells! Total assay time: Kit size options: |
Mabtech
The Swedish biotech company Mabtech emerged from the Department of Immunology at Stockholm University in 1986. Mabtech develops and manufactures high quality monoclonal antibodies and kits for ELISA, ELISpot and FluoroSpot and is a world leader in advancing the ELISpot and FluoroSpot methods.
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