FluoroSpot is an essential immune profiling assay to quantify cellular responses by detecting the frequency of cytokine secreting cells. The FluoroSpot assay exhibits superior sensitivity, making it a great solution for detecting rare antigen-specific cells and understanding complex immune responses. 

FluoroSpot assays are particularly relevant for several therapeutic areas including:

• Infectious disease research: Identify and quantify antigen-specific cytokine secretion. Understand the immune mechanisms behind Tuberculosis, influenza, SARS-CoV-2, HIV, Epstein-Barr virus, and more. 
• Vaccine development: Assess vaccine efficacy by measuring antigen-specific cytokine responses before and after immunization. FluoroSpot helps identify whether your vaccine generates the right type of immune response, for example, detecting cells that co-produce IFN-γ and TNF-α indicates strong Th1 immunity. 
• Cancer immunotherapy: Monitor tumor-specific T cell responses to checkpoint inhibitors, CAR-T therapies, or cancer vaccines. Track whether treatment enhances anti-tumor cytokine production (IFN-γ/TNF-α) from tumor antigen-specific cells and identify patients who might benefit from combination therapies based on their cytokine profiles.
• Allergy: Distinguish between different T helper cell responses to allergens. Measure IL-6 production alongside other cytokines to understand allergic responses, or track how immunotherapy treatments shift cytokine profiles from pro-allergic to tolerogenic patterns in allergen-specific T cells.
• Autoimmunity: Identify self-reactive T cells producing pathogenic cytokine combinations. In multiple sclerosis research, detect myelin-specific cells co-producing IFN-γ and TNF-α. Monitor how immunosuppressive treatments affect the frequency and cytokine profile of autoreactive cells, helping optimize therapeutic approaches.

How do ELISpot and FluoroSpot compare to ELISA?

1. Clear-bottom microwell plates are coated with a mixture of capture antibodies specific to the analytes of interest and incubated overnight for 16-18 hours. Our FluoroSpot MAX Deluxe sets contain pre-optimized concentrations of capture antibodies specific to each assay. 
2. The following day, capture antibodies are washed away, and cells of interest are added to the wells, along with any stimuli (such as specific antigens or polyclonal cell activators like our Cell Activation Cocktail (without Brefeldin A). We suggest running all conditions in triplicate wells. 
3. During the incubation period, plate-bound capture antibodies surrounding the cells capture the analytes of interest directly at the source of secretion. The incubation time should be optimized to account for the kinetics of each analyte’s secretion and stimulation. 
4. After the incubation period, cells are washed away from the plate and fluorophore-conjugated detection antibodies are added to the plate where they form complexes with the plate-immobilized analytes. The plate is washed and subsequently imaged using an image cytometer or similar fluorescence imaging system to visualize individual spots. Fluorescent spots represent the “footprint” of analyte secreting cells within the plate. 

An overview of the assay procedure for FluoroSpot MAX Deluxe assays.

Streamlined workflow, superior results

Our FluoroSpot MAX Deluxe Sets take a unique approach to the traditional FluoroSpot assay, which makes the workflow more streamlined and accessible. 

Clear-bottom imaging plates: Unlike traditional PVDF membrane plates that require ethanol activation, manual underdrain removal, and lengthy drying times, FluoroSpot MAX Deluxe sets contain pre-optimized reagents compatible with clear-bottom imaging plates to eliminate these tedious and time-consuming steps. You can image directly after spot development, reducing your assay time significantly without sacrificing assay performance. We suggest using a black-walled, clear-bottom, high-binding imaging plate such as Corning Cat. No 3601 or equivalent plate. 

We ensured the performance of our FluoroSpot MAX Deluxe Set utilizing clear-bottom plates is comparable to the equivalent ELISpot assay using PVDF membrane plates by running both assays in parallel. Clear-bottom plates exhibited no noticeable differences in spot distribution when compared to the equivalent ELISpot assay using PVDF plates. 

10,000 PBMC were stimulated with 1X Cell Activation Cocktail without Brefeldin A, and FluoroSpot MAX assay (clear-bottom plate, top) or ELISpot MAX (PVDF plate, bottom) was run in parallel for Human IFN-γ (Cat. No. 430124, 430126), Human TNF-α (Cat. No. 430224, 430226), and Human IL-6 (Cat. No. 431324, 430523). Images are a representation of triplicate assay wells.

Flexible instrumentation: Because a clear-bottom imaging plate is used in the FluoroSpot MAX assay, there is no longer a need for single-purpose, dedicated FluoroSpot readers. FluoroSpot MAX Deluxe is compatible with imaging cytometers and other fluorescence imaging platforms capable of whole-well fluorescence imaging, making this powerful technology accessible to more labs without access to traditional FluoroSpot readers. 

Explore our comprehensive user guide for FluoroSpot MAX Deluxe analysis using the Revvity Celigo™ Image Cytometer.

Available FluoroSpot MAX Deluxe Sets

Explore all of our FluoroSpot MAX Deluxe Sets. Each kit includes capture antibodies, detection antibodies, positive controls, and all necessary buffers – everything you need except the plates and antigens. 

Don’t see your analyte of interest? Get in touch with us to let us know how we can help.