Human tumor necrosis factor-alpha (TNF-α) ELISpot Kit is designed for the quantitative detection and enumeration of cells secreting human TNF-α in a suspension of peripheral blood mononuclear cells (PBMCs). ELISpot is commonly used for evaluating specific immune responses, such as determining the frequency of activated T cell subsets in response to antigenic stimulation. TNF-α is secreted by many cell types including activated CD8+ cytotoxic T cells and the T helper (Th) subsets Th1, Th2, Th17, Th9, and Th22. ELISpot is highly sensitive and can detect secreted analytes at a single-cell level. This assay is used in research of infectious diseases, cancers, allergies, and autoimmune diseases.
The assay is based on a modified version of the sandwich ELISA method, in which cells are added to ELISpot plates pre-coated with capture antibodies specific for the cytokine. The cytokine is then captured throughout the culture period and is detected by the addition of a biotinylated detection antibody, followed by streptavidin-alkaline phosphatase (SA-ALP), which binds to the biotinylated antibody. Addition of the chromogenic enzyme substrate 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP)/nitroblue tetrazolium (NBT), results in the formation of colored spots. The number, size, and intensity of these spots are directly proportional to the frequency of cytokine-secreting cells in the sample and the amount of analyte secreted.
Figure 1. Human TNF-alpha ELISpot Kit Detects TNF-alpha-Secreting T Cells Following Polyclonal Stimulation.
ELISpot analysis of human peripheral blood mononuclear cells (PBMCs) isolated from whole blood using Lymphoprep™ (Catalog #18060) density gradient medium or EasySep™ Direct Human PBMC Isolation Kit (Catalog #19654). Cells were cultured in ImmunoCult™-XF T Cell Expansion Medium (Catalog #10981) in the absence of stimuli (negative control) or presence of PMA (Catalog #74044) + Ionomycin (Catalog #73724). Cells were seeded at 1 x 10^4 cells/well on Human TNF-α pre-coated white plate and incubated at 37°C, 5% CO2 for 18 hours. Following incubation, ELISpot plates were processed as per the instructions on the Product Information Sheet. Wells were imaged and counted using an ELISpot reader.
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