Description
Human IL-10 ELISpot Kit
Assay Genie ELISpot is a highly specific immunoassay for the analysis of IL-10 production and secretion from T-cells at a single cell level in conditions closely comparable to the in-vivo environment with minimal cell manipulation. This technique is designed to determine the frequency of IL-10 producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. Utilising sandwich immuno-enzyme technology, Assay Genie ELISpot assays can detect both secreted IL-10 (qualitative analysis) and single cells that produce IL-10 (quantitative analysis). Cell secreted IL-10 is captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured IL-10 is revealed by tracer antibodies and appropriate conjugates.
| Product type: | ELISpot Kit |
| Size: | 10 x 96 Assays |
| Target species: | Human |
| Specificity: | Recognizes natural human IL-10 |
| Incubation: | 3h after cell stimulation |
| Kit content: | Assay Genie Pre-coated ELISpot kits include precoated PVDF plates, Detection antibody, Alkaline phosphatase conjugate, BSA, BCIP/NBT ready-to-use substrate buffer. |
| Synonyms: | N/A |
| Uniprot: | P22301 |
A capture antibody highly specific for IL-10 is 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 minimise 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 IL-10 produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured IL-10. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing, substrate is then applied to the wells resulting in coloured spots which can be quantified using appropriate analysis software or manually using a microscope.
| Step | Procedure |
| 1. | For PVDF membrane activation, add 25 µl of 35% ethanol to every well. |
| 2. | Incubate plate at room temperature (RT) for 30 seconds. |
| 3. | Empty the wells by flicking the plate over a sink & gently tapping on absorbent paper. Thoroughly wash the plate 3x with 100 µl of PBS 1X per well. |
| 4. | Add 100 µl of diluted capture antibody to every well. |
| 5. | Cover the plate and incubate at 4°C overnight. |
| 6. | Empty the wells as previous and wash the plate once with 100 µl of PBS 1X per well. |
| 7. | Add 100 µl of blocking buffer to every well. |
| 8. | Cover the plate and incubate at RT for 2 hours. |
| 9. | Empty the wells as previous and thoroughly wash once with 100 µl of PBS 1X per well. |
| 10. | Add 100 µl of sample, positive and negative controls cell suspension to appropriate wells providing the required concentration of cells and stimulant. |
| 11. | Cover the plate and incubate at 37°C in a CO2 incubator for an appropriate length of time (15-20 hours). Note: do not agitate or move the plate during this incubation. |
| 12. | Empty the wells and remove excess solution then add 100 µl of Wash Buffer to every well. |
| 13. | Incubate the plate at 4°C for 10 min. |
| 14. | Empty the wells as previous and wash the plate 3x with 100 µl of Wash Buffer. |
| 15. | Add 100 µl of diluted detection antibody to every well. |
| 16. | Cover the plate and incubate at RT for 1 hour 30 min. |
| 17. | Empty the wells as previous and wash the plate 3x with 100 µl of Wash Buffer. |
| 18. | Add 100 µl of diluted Streptavidin-conjugate to every well. |
| 19. | Cover the plate and incubate at RT following the supplier's instructions. |
| 20. | Empty the wells and wash the plate 3x with 100 µl of Wash Buffer. |
| 21. | Peel of the plate bottom and wash both sides of the membrane 3x under running distilled water, once washing complete remove any excess solution by repeated tapping on absorbent paper. |
| 22. | Add 100 µl of ready-to-use substrate buffer to every well. |
| 23. | Following the supplier's instructions, incubate the plate for 5-15 min monitoring spot formation visually throughout the incubation period to assess sufficient colour development. |
| 24. | Empty the wells and rinse both sides of the membrane 3x under running distilled water. Completely remove any excess solution by gentle repeated tapping on absorbent paper Read Spots: allow the wells to dry and then read results. The frequency of the resulting coloured spots. |
| UniProt Protein Function: | IL10: Inhibits the synthesis of a number of cytokines, including IFN-gamma, IL-2, IL-3, TNF and GM-CSF produced by activated macrophages and by helper T-cells. Belongs to the IL-10 family. |
| UniProt Protein Details: | Protein type:Motility/polarity/chemotaxis; Cytokine; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 1q31-q32 Cellular Component: extracellular space Molecular Function:growth factor activity; cytokine activity; interleukin-10 receptor binding Biological Process: B cell proliferation; negative regulation of chronic inflammatory response to antigenic stimulus; regulation of isotype switching; positive regulation of transcription, DNA-dependent; leukocyte chemotaxis; response to glucocorticoid stimulus; positive regulation of JAK-STAT cascade; negative regulation of B cell proliferation; negative regulation of membrane protein ectodomain proteolysis; response to insulin stimulus; negative regulation of interferon-alpha biosynthetic process; negative regulation of T cell proliferation; cytoplasmic sequestering of NF-kappaB; cell-cell signaling; positive regulation of MHC class II biosynthetic process; negative regulation of interferon-gamma production; response to molecule of bacterial origin; negative regulation of interleukin-6 production; hemopoiesis; negative regulation of tumor necrosis factor biosynthetic process; inflammatory response; aging; response to drug; response to inactivity; negative regulation of interleukin-12 production; positive regulation of B cell apoptosis; T-helper 2 type immune response; negative regulation of myeloid dendritic cell activation; negative regulation of nitric oxide biosynthetic process; negative regulation of cytokine secretion during immune response; regulation of gene expression; B cell differentiation; defense response to bacterium; negative regulation of MHC class II biosynthetic process; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription factor activity; receptor biosynthetic process; response to activity; regulation of sensory perception of pain; negative regulation of apoptosis; positive regulation of cytokine secretion Disease: Graft-versus-host Disease, Susceptibility To; Rheumatoid Arthritis; Human Immunodeficiency Virus Type 1, Susceptibility To |
| NCBI Summary: | The protein encoded by this gene is a cytokine produced primarily by monocytes and to a lesser extent by lymphocytes. This cytokine has pleiotropic effects in immunoregulation and inflammation. It down-regulates the expression of Th1 cytokines, MHC class II Ags, and costimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. This cytokine can block NF-kappa B activity, and is involved in the regulation of the JAK-STAT signaling pathway. Knockout studies in mice suggested the function of this cytokine as an essential immunoregulator in the intestinal tract. Mutations in this gene are associated with an increased susceptibility to HIV-1 infection and rheumatoid arthritis.[provided by RefSeq, May 2011] |
| UniProt Code: | P22301 |
| NCBI GenInfo Identifier: | 124292 |
| NCBI Gene ID: | 3586 |
| NCBI Accession: | P22301.1 |
| UniProt Related Accession: | P22301 |
| Molecular Weight: | 20,517 Da |
| NCBI Full Name: | Interleukin-10 |
| NCBI Synonym Full Names: | interleukin 10 |
| NCBI Official Symbol: | IL10Â Â |
| NCBI Official Synonym Symbols: | CSIF; TGIF; GVHDS; IL-10; IL10AÂ Â |
| NCBI Protein Information: | interleukin-10; T-cell growth inhibitory factor; cytokine synthesis inhibitory factor |
| UniProt Protein Name: | Interleukin-10 |
| UniProt Synonym Protein Names: | Cytokine synthesis inhibitory factor; CSIF |
| Protein Family: | Interleukin |
| UniProt Gene Name: | IL10Â Â |
| UniProt Entry Name: | IL10_HUMAN |
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