Mouse Interferon-γ (IFN-γ) ELISPOT Kit

背景（Background）

Interferon-γ (IFN-γ) is the only type II interferon. This proinflammatory cytokine is secreted by activated T cells and NK cells. It activates macrophages and endothelial cells and regulates immune responses by affecting APCs, T cells, and B cells. Production of IFN-γ by helper T cells and cytotoxic T cells is a hallmark of the Th1-type phenotype. Thus, high-level production of IFN-γ is typically associated with effective host defense against intracellular pathogens. The Mouse IFN-γ ELISpot assay is designed for the detection of mouse IFN-γ secreting cells at the single cell level and can be used to quantitate the frequency of mouse IFN-γ secreting cells. ELISpot assays are highly reproducible and sensitive, and can be used to measure responses with frequencies well below 1 in 100,000. ELISpot assays do not require prior in vitro expansion of T cells and they are suitable for high-throughput analysis using only small volumes of primary cells. As such, ELISpot assays are useful tools for research in vaccine development, cytokine secretion and the monitoring of various clinical trials.

应用说明（Application）

This kit is used to detect IFN-γ at the cellular level.

It is for research use only.

存储（Storage）

1. Unopened kit should be stored at 2℃-8℃ upon receiving.

2. Find the expiration date on the outside packaging and do not use reagents past their expiration date.

3. Please use it up once you opened it.

原理（Assay Principles）

ELISpot assays employ the quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. The Kit consists of Pre-coated Anti-IFN-γ Antibody Microplate, Positive Stimulus, Biotin-Anti-IFN-γ Antibody, Streptavidin-HRP, AEC and buffers.

Your experiment will include 6 simple steps:

a) A monoclonal antibody specific for mouse IFN-γ has been pre-coated onto a polyvinylidene difluoride (PVDF)-backed microplate.

b) Appropriately stimulated cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO2 incubator for a specified period of time.During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IFN-γ.

c) After washing away any cells and unbound substances, a biotinylated antibody specific for mouse IFN-γ is added to the wells. Following a wash to remove any unbound biotinylated antibody, HRP conjugated to streptavidin is added.

d) Wash the plate and add the Streptavidin-HRP diluted by Dilution Buffer to the plate.

e) Wash the plate and add AEC. A reddish-brown colored precipitate forms and appears as spots at the sites of cytokine localization, with each individual spot representing an individual IFN-γ secreting cell.

f) The spots can be counted with an automated ELISpot reader system or manually using a stereomicroscope.