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- Ultra-sensitive: detection of a single cytokine-secreting cell in a million
- Unique: analysis of viable cells at single-cell level
- Ground-breaking: optional magnetic enrichment for multiparametric phenotyping of viable cells
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| Overview |
| The IFN-γ Secretion Assay was developed for the sensitive detection of human IFN-γ-secreting cells. |
| Details |
Background information
IFN-γ (interferon-gamma) is predominantly secreted by activated CD8+ and CD4+ memory and effector T cells and by NK cells. It is mainly involved in the regulation of inflammatory immune responses. These TH1 types of immune mechanisms are effective against intracellular pathogens and tumors. IFN-γ-secreting T cells can also be involved in immunological disorders, such as autoimmune reactions.
Downstream applications
Virus-specific T cells were investigated after stimulation with peptides or proteins derived from influenza virus1, CMV2,3, EBV4,5,6, HIV5,7,8,9,10, HBV11, and ADV24.
Virus-specific T cells were expanded in vitro1,2,3,5,6,9 showing highly specific and very efficient killing of target cells and have been analyzed for TCR clonotypes8,10.
The IFN-γ Secretion Assay was used for the isolation and analysis of antigen-specific T cells from PBMCs after stimulation with Tetanus Toxoid1, minor histocompatibility antigens, and tumor antigens12,13,14,15. The assay was also used to purify and analyze tumor-specific T cells from T cell lines13,14, for the isolation of functional antigen-specific, IFN-γ-secreting T cells reacting to other tumor antigens, e.g. SSX14, CEA16, or HER217 from PBMCs or TILs (tumor infiltrating lymphocytes)14,18.
The IFN-γ Secretion Assay was used to counterstain peptide-MHC-tetramer-labeled Melan A-specific CD8+ T cells to analyze functionality of the tetramer-positive cells.12,15
The IFN-γ Secretion Assay was also used for isolation and functional characterization of allergen-specific T cells.21 Furthermore, the IFN-γ Secretion Assay was used for epitopemapping of MHC class II peptides.19
IFN-γ-secreting human NK cells were isolated using the IFN-γ Secretion Assay.20 IFN-γ Secretion Assay reagents were reported to cross-react with chimpanzee cells22 but not with rhesus macaque cells.
The IFN-γ Secretion Assay can also be used for two-color cytokine analysis9 and allows counterstaining of peptide- MHC-tetramer-labeled cells 12,15. It can also be combined with flow cytometric proliferation assays 23. |
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| Figure 1 |
PBMCs of a CMV+ donor were stimulated for 16 hours with CMV lysate. The responding cells were stained and isolated according to secretion of IFN-γ using the IFN-γ Secretion Assay - Cell Enrichment and Detection Kit.
In the stimulated sample, 1292 IFN-γ-secreting CD4+ T cells were enriched per 106 CD4+ T cells using MS Columns and a MiniMACS™ Separator. In the unstimulated control sample, no IFN-γ-secreting CD4+ T cell were enriched per 106 CD4+ T cells. |
| Stimulated sample |
| A: Before enrichment |
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| B: After enrichment |
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| Unstimulated control |
| C: Before enrichment |
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| D: After enrichment |
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* Percentage represents frequency among CD4+ T cells.
** Percentage represents frequency among enriched cells. |
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| Products |
| IFN-γ Secretion Assay – Detection Kit (PE), human |
- for 100 tests with 106 total cells
Download datasheet
130-054-202
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| IFN-γ Secretion Assay – Detection Kit (FITC), human |
- for 100 tests with 106 total cells
Download datasheet
130-090-433
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| IFN-γ Secretion Assay – Detection Kit (APC), human |
- for 100 tests with 106 total cells
Download datasheet
130-090-762
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| References |
| 1. Brosterhus et al. (1999) Eur. J. Immunol. 29: 4053-4059. |
| 2. Bissinger et al. (2002) Exp.Hematol. 30: 1178-1184. |
| 3. Bitmansour et al. (2002) J. Immonol.169: 1207-1218. |
| 4. Bickham et al. (2001) J. Clin. Invest. 107: 121-130. |
| 5. Cohen et al. (2002) Virology 304: 474-484. |
| 6. Koehne et al. (2002) Blood 99: 1730-1740. |
| 7. Altfeld et al. (2001) J. Immunol. 167: 2743-2752. |
| 8. Douek et al. (2002) Nature 417: 95-98. |
| 9. Lichterfeld et al. (2004) Blood 104: 487-494. |
| 10. Lee et al. (2002) J. Clin. Invest. 110: 1339-1347. |
| 11. Desombre et al. (2003) J. Immunol. Meth. 286: 167-185. |
| 12. Meidenbauer et al. (2003) J. Immunol. 170: 2161-2169. |
| 13. Oelke et al. (2000) Clin. Cancer Res. 6: 1997-2005. |
| 14. Ayyoub et al. (2004) J. Clin. Invest. 113: 1225-1233. |
| 15. Pittet et al. (2001) J. Immunol. 166: 7634-7640. |
| 16. Maerten et al. (2002) Cancer Immunol. Immunother. 51: 25-32. |
| 17. Meyer zu Büschenfelde et al. (2001) J. Immunol. 167: 1712-1719. |
| 18. Becker et al. (2001) Nature Med. 7: 1159-1162. |
| 19. Novak et al. (2001) J. Immunol. 166: 6665-6670. |
| 20. Deniz et al. (2002) Eur. J. Immunol. 32: 879-884. |
| 21. Akdis et al. (2004) J. Exp. Med. 199: 1567-1575. |
| 22. Meyer-Olson et al. (2003) J. Immunol. 170: 4161-4169. |
| 23. Gutzmer et al. (2003) J. Immunol. 171: 6363-6371. |
| 24. Feuchtinger et al. (2004) Exp. Hematol. 32: 282-289. |
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