Dual-color FluoroSpot assay. In green IFN-gamma; in red IL-10;
in yellow IFN-gamma/IL-10
Stimulation: PMA-ionomycin
HUMAN Cytokine IFN-Gamma/IL-10 FluoroSpot kit
INTENDED USE
The dual-color cytokine FluoroSpot assay is a modification of the Enzyme-Linked ImmunoSPOT (ELISPOT) assay and is designed to detect double cytokine-secreting T cells at the single cell level. The assay is based on the use of fluorescent- instead of enzymatic-labeled conjugates. Previous attempts to develop an immunoenzymatic dual-color ELISPOT assay were less successful because of problematic interpretation of mixed color spots. The dual-color FluoroSpot assay, however, provides good discrimination between single and double cytokine-secreting cells and is particularly suited to detect T cell subpopulations with a characteristic cytokine profile. The sensitivity of the assay lends itself to measure very low frequencies of cytokine-secreting T cells (1/300,000).
BRIEF DESCRIPTION DUAL 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 one cytokine of interest whereas yellow spots originate from cells that release both cytokines of interest.
Contents:
- Human IFN-gamma/IL-10 FluoroSpot kit:
- Coating antibodies
- Biotinylated detector antibodies
- Fluorescent-labeled conjugate
- Blocking stock solution R
- Dilution buffer R
- Tween-20
- PVDF FluoroSpot plates with lids and cover slips
- Detailed manual
Streamline laboratory procedures! Click here for ready-to-use reagents and buffers, from Medicago AB. Available in tablets and pouches!
