Description
Key facts
Sample types:Suspension cells,
Reacts with:Human,
Assay type:Sandwich (qualitative),
Assay Platform:Reagents
Product details:
The ELISPOT assay is designed to enumerate cytokine producing cells in a single cell suspension. This method has the advantage of requiring a minimum of in-vitro manipulations allowing cytokine production analysis as close as possible to in-vivo conditions in a highly specific way. This technique is designed to determine the frequency of cytokine producing cells under a given stimulation, and the follow-up of such frequency during a treatment and/or a pathological state. ELISPOT assay constitutes an ideal tool in the TH1 / TH2 response, vaccine development, viral infection monitoring and treatment, oncology, infectious diseases, autoimmune diseases and transplantation.
The ELISPOT assay is based on sandwich immuno-enzyme technology. Cell secreted cytokines or soluble molecules are captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured cytokines are revealed by tracer antibodies and appropriate conjugates.
The dual colour ELISPOT allows you to monitor the production of two cytokines simultaneously in the same well.
Principle
After cell stimulation, locally produced cytokines are captured by IFN gamma and IL10 specific monoclonal antibodies. After cell lysis, trapped cytokine molecules are revealed by a secondary anti-IFN gamma FITC conjugated antibody and a biotinylated anti-IL-10 antibody. Those are in turn recognised by anti-FITC HRP and streptavidin-AP conjugates. PVDF-bottomed-well plates are then incubated first with AEC substrate buffer, washed and subsequently incubated with BCIP/NBT. Coloured red/brownish spots indicate IFN gamma production while IL-10 is revealed by blue/purple spots.
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) and interleukin 10 (IL-10) are critical cytokines in immune regulation. IFN-γ also known as type II interferon weighs approximately 16-17 kDa and is predominantly expressed by T cells and natural killer cells. IL-10 a cytokine synthesis inhibitory factor has a similar mass of about 18 kDa and is expressed by regulatory T cells macrophages and dendritic cells. Both cytokines are located in tissues involved in immune responses such as lymphoid tissues.
Biological function summary
IFN-γ activates macrophages and increases antigen presentation enhancing the immune response. IL-10 functions oppositely suppressing inflammation and limiting immune responses to prevent tissue damage. These cytokines do not form a complex together but act synergistically in regulating immune responses. They maintain a balanced immune system by coordinating pro-inflammatory and anti-inflammatory signals.
Pathways
IFN-γ is central in the JAK-STAT pathway influencing cellular responses and gene expression. IL-10 modulates the MAPK pathway contributing to its anti-inflammatory activities. Both pathways are important for immune homeostasis with IFN-γ closely interacting with proteins like STAT1 and IL-10 modulating responses by impacting proteins such as IL-12.
An imbalance between IFN-γ and IL-10 associates with autoimmune conditions and chronic infections. Excessive IFN-γ can lead to autoimmunity where the balance with IL-10 becomes disrupted. Diseases like rheumatoid arthritis and tuberculosis show involvement of these cytokines with IL-10 often attempting to counteract the immune system's hyperactivation. Other related proteins like TNF-α also engage in these pathological processes further complicating disease outcomes.
