Description
Key facts
Detection method:Colorimetric,
Sample types:Suspension cells,
Reacts with:Mouse,
Assay type:Sandwich (qualitative),
Assay Platform:Microplate
Product details:
Mouse Interferon gamma ELISPOT Kit ab64029 is a sensitive assay for the qualitative measurement of mouse IFN gamma production and secretion in a single cell suspension
How the assay works
The ELISPOT assay involves a capture antibody highly specific for the analyte of interest coated to the wells of a PVDF bottomed 96 well microtitre plate, either during kit manufacture or in the laboratory. The plate is then blocked to minimize any non-antibody dependent unspecific binding and washed. Cell suspension and stimulant are added and the plate incubated allowing the specific antibodies to bind any analytes produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured analyte. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing substrate is then applied to the wells resulting in colored spots which can be quantified using appropriate analysis software or manually using a microscope.
Mouse Interferon gamma ELISPOT Kit ab64029 protocol summary:
1. Add sample (Cells) to appropriate wells. Incubate at 37 C.
2. Aspirate and wash each well. Add prepared Biotinylated labeled detector antibody. Incubate at room temperature.
3. Add prepared Streptavidin-Alkaline Phosphatase mix to each well. Incubate at room temperature.
4. Add the substrate solution BCIP/NBT to each well and monitor spot formation.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions-+4°C, Appropriate long-term storage conditions-+4°C, Storage information-+4°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Interferon gamma (IFN-γ) also known as type II interferon is a cytokine that plays an important role in immune response. IFN-γ has a molecular weight of about 17 kDa and is produced by T cells and natural killer (NK) cells. IFN-γ binds to the interferon gamma receptor initiating a signaling cascade that activates various genes involved in immune functions. It is expressed mainly in activated immune cells within lymphoid tissues and inflamed sites during immune responses.
Biological function summary
This cytokine is significant in promoting macrophage activation enhancing the antigen presentation process and boosting the antimicrobial activity of phagocytes. IFN-γ is not part of a larger protein complex but works as a homodimer in signal transduction. Its production heightens the Th1 immune response by stimulating the differentiation of naïve T cells into Th1 cells which is essential for effective cellular immunity.
Pathways
IFN-γ is integrally involved in the JAK-STAT signaling pathway alongside another critical cytokine Interleukin-12. This pathway further amplifies the immune response by regulating the expression of genes associated with cellular defense mechanisms. IFN-γ also interacts with the NF-kB pathway influencing inflammation and the activation of further immune responses. These interactions show a network of cooperativity with proteins like STAT1 and NF-kB essential for executing its biological roles.
IFN-γ is linked to autoimmune diseases such as rheumatoid arthritis and multiple sclerosis where its elevated levels can exacerbate inflammatory processes. It connects to other proteins like TNF-alpha in promoting the inflammatory cascade. Moreover lower levels of IFN-γ are associated with a heightened risk of infections like tuberculosis demonstrating its vital role in pathogen defense. Therefore understanding IFN-γ and its interactions can be key in developing therapeutic approaches against these conditions.
