* Following cell stimulation.
ELISpot kits are highly sensitive, microplate-based assays for the detection of cytokine secreting cells. This kit is designed for the simultaneous detection and enumeration of IFN-γ/IL-5. Complete ELISpot kits are ready-to-run and require no assay development or refinement.
Appropriately stimulated cells are pipetted directly into the wells of a PVDF-backed microplate. The PVDF membrane has been pre-coated with capture antibodies specific for IFN-γ/IL-5. The microplate is then placed in a humidified 37 °C CO2 incubator for the desired period of time. During this incubation period, the immobilized capture antibodies in the immediate vicinity of the secreting cells bind secreted IFN-γ/IL-5.
At the desired time point, one analyte is detected using a biotinylated detection antibody and alkaline-phosphatase (AP)-conjugated to streptavidin. The second analyte is detected using a horseradish peroxidase (HRP)-conjugated detection antibody. Following incubation with the chromogens BCIP/NBT (substrate for AP, blue-black precipitate) and AEC (substrate for HRP, red precipitate), spots form to indicate the presence of IFN-γ/IL-5 secreting cells. Each spot represents an individual secreting cell. The spots can be counted with an ELISpot reader system or using a stereomicroscope.
OTHER REAGENTS REQUIRED
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