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Your Position: > Kits > IFN-gamma > RAS-SP002

Mouse Interferon-γ (IFN-γ) ELISPOT Kit

For research use only.
    Materials Provided
    IDComponentsSize
    RSP002-C01Pre-coated Anti-IFN-γ Antibody Microplate1 plate
    RSP002-C02Positive Stimulus60 μg
    RSP002-C03Biotin-Anti-IFN-γ Antibody50 μL
    RSP002-C04Streptavidin-HRP50 μL
    RSP002-C05Washing Buffer (10x)50 mL
    RSP002-C06Dilution Buffer (1x)50 mL
    RSP002-C07AEC Dilution25 mL
    RSP002-C08AEC Solution A (20x)0.8 mL
    RSP002-C09AEC Solution B (20x)0.8 mL
    RSP002-C10AEC Solution C (20x)0.8 mL
  • 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.
  • Application

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

    It is for research use only.

  • 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.

  • 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.

Typical Data Please refer to DS document for the assay protocol.
 IFN-gamma TYPICAL DATA

1x10⁵ cells/well Mouse Spleen Cells were stimulated with 6.0 μg/mL Positive Stimulus and cultured at 37℃ and 5%CO2 for 20 h, 308 spots were produced. The following example data is for reference only.

  • Clinical and Translational Updates

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