FluoroSpot Assays: A Multiplex Evolution of ELISpot

FluoroSpot assays build on the principles of ELISpot, but introduce multiplexing capabilities through fluorescence-based detection. This allows researchers to simultaneously detect multiple analytes secreted by the same or different cells within a single well, providing a more comprehensive view of antigen-specific immune responses.

Our FluoroSpot MAX™ Deluxe Sets were designed to overcome challenges with classical PVDF membrane-based FluoroSpot assays and utilize fluorophore-conjugated detection antibodies for fast one-step detection. FluoroSpot MAX Deluxe Sets may be imaged using an image cytometer or other imaging platform capable of whole-well imaging. The device should be equipped with the appropriate filters for the fluorophores utilized in each assay.  

• Multiplex detection: Save time and resources by simultaneously detecting multiple cytokines or analytes in one assay.
• Enhanced data insights: Gain detailed information about polyfunctional cells secreting multiple cytokines, a critical aspect in understanding complex antigen-specific immune responses.
• Streamlined workflow: Reduce assay time by multiplexing with fluorophore-conjugated detection antibodies and one-step detection. Clear-bottom plates remove the need for underdrain removal and long drying times. 
• High sensitivity and specificity: Retain the single-cell resolution and sensitivity of ELISpot while expanding analytical power.

How FluoroSpot Works

Instead of enzymatic reactions, FluoroSpot employs fluorophore-labeled detection antibodies to identify secreted analytes. The use of distinct fluorophores enables the simultaneous detection of multiple targets in different fluorescent channels, such as APC and PE. Each fluorescent spot represents a specific analyte secreted by an individual cell. Single- and multi-analyte secreting cells can then be quantified with the FluoroSpot assay.  

FluoroSpot MAX Deluxe Set Human TNF-α/IFN-γ (Cat. No. 430228)

Human PBMCs (5,000 cells/well) were incubated for 5 hours in the presence of 1X Cell Activation Cocktail without Brefeldin A (Cat. No. 423301). Our Cell Activation cocktail contains PMA and ionomycin at optimized concentrations. The number of cells secreting Human TNF-α/ IFN-γ was analyzed by FluoroSpot and quantified using an image cytometer.

Not Ready to Multiplex? 

FluoroSpot MAX Deluxe Sets are also available as single analyte assays for focusing on specific targets. FluoroSpot MAX Deluxe single analyte assays utilize a two-step detection method consisting of biotinylated detection antibodies plus streptavidin-PE, resulting in fluorescent “footprints” of analyte secreting cells directly in clear-bottom microplates.

FluoroSpot MAX Deluxe Sets contain pre-optimized reagents including antibodies, buffers and solutions, while still being a cost-effective option. Clear-bottom microplates are not provided. 

FluoroSpot MAX Deluxe Assay Imaging and Analysis

To perform imaging and analysis of FluoroSpot MAX Deluxe assay plates using an Image Cytometer, please refer to the corresponding User Guide for compatible instruments.

Celigo Image Cytometer User Guide