FluoroSpot
The bright way to detect cells secreting multiple cytokines.
The Fluorospot assay is in principle based on ELISpot but utilizes fluorescent-based detection systems, enabling the detection of cells secreting either of two different cytokines, or both, in the same well.
Fluorospot is in particular suitable for analysis of polyfunctional T cells, defined by their secretion of multiple cytokines, that have been suggested to be of high relevance for protection against infectious diseases e.g. HIV.
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The FluoroSpot procedure
(1) A mixture of two cytokine-specific antibodies (Ab) is immobilized on a 96-well IPFL plate (designed for low autofluorescense).
(2) Cells are added in the presence or absence of specific stimulus.
(3) During incubation the cells become activated by the stimulus and start to produce and secrete cytokines that bind to the capture Abs.
(4) Cells are removed and a mixture of two different anti-cytokine detection Abs, labeled with FITC and biotin respectively, is added.
(5) A mixture of anti-FITC-green and Streptavidin-red is added.
(6) After incubation, a fluorescence enhancer solution is added.
(7) Finally, the plate is analyzed in a FluoroSpot reader. A reader that can detect co-localized green and red spots, i.e. dual secreting cells, is preferentially used. Co-localized spots are shown graphically as yellow in an image overlay. By counting the number of spots in stimulated cultures and controls without stimulus the frequency of responding singe and double secreting cells is determined.
 Download our FluoroSpot Brochure as a PDF
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