The IFN-γ Secretion Assay - Cell Enrichment and Detection Kit was developed for the sensitive detection as well as the enrichment of human IFN-γ-secreting cells.
The used Anti-PE MicroBeads UltraPure enhances sensitivity and minimizes background of debris and dead cells.

Data and images for IFN-γ Secretion Assay – Cell Enrichment and Detection Kit (PE), human

Figures

Figure 1

PBMCs from a CMV
+
donor were stimulated with PepTivator™ CMV pp65 or left untreated. Cells were stained and isolated according to secretion of IFN-γ using the IFN-γ Secretion Assay - Cell Enrichment and Detection Kit. The cells were counterstained with CD8-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.
Stimulated sample
Before enrichment
After enrichment
View details

Figure 1

PBMCs from a CMV
+
donor were stimulated with PepTivator™ CMV pp65 or left untreated. Cells were stained and isolated according to secretion of IFN-γ using the IFN-γ Secretion Assay - Cell Enrichment and Detection Kit. The cells were counterstained with CD8-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.
View details

Figure 1

PBMCs from a CMV
+
donor were stimulated with PepTivator™ CMV pp65 or left untreated. Cells were stained and isolated according to secretion of IFN-γ using the IFN-γ Secretion Assay - Cell Enrichment and Detection Kit. The cells were counterstained with CD8-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.
Unstimulated control
Before enrichment
After enrichment
View details

Figure 1

PBMCs from a CMV
+
donor were stimulated with PepTivator™ CMV pp65 or left untreated. Cells were stained and isolated according to secretion of IFN-γ using the IFN-γ Secretion Assay - Cell Enrichment and Detection Kit. The cells were counterstained with CD8-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.
View details

Figure 1

PBMCs from a CMV
+
donor were stimulated with PepTivator™ CMV pp65 or left untreated. Cells were stained and isolated according to secretion of IFN-γ using the IFN-γ Secretion Assay - Cell Enrichment and Detection Kit. The cells were counterstained with CD8-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and PI fluorescence.

Specifications for IFN-γ Secretion Assay – Cell Enrichment and Detection Kit (PE), human

Overview

The IFN-γ Secretion Assay - Cell Enrichment and Detection Kit was developed for the sensitive detection as well as the enrichment of human IFN-γ-secreting cells.
The used Anti-PE MicroBeads UltraPure enhances sensitivity and minimizes background of debris and dead cells.

Detailed product information

Background information

Interferon gamma (IFN-γ) 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 T1 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 virus
1
, CMV
2,3
, EBV
4,5,6
, HIV
5,7,8,9,10
, HBV
11
, and ADV
24
.
Virus-specific T cells were expanded
invitro1,2,3,5,6,9
showing highly specific and very efficient killing of target cells and have been analyzed for TCR clonotypes
8,10
.
IFN-γ Secretion Assays were used for the isolation and analysis of antigen-specific T cells from PBMCs after stimulation with Tetanus Toxoid
1
, minor histocompatibility antigens, and tumor antigens
12,13,14,15
. IFN-γ Secretion Assays were also used to purify and analyze tumor-specific T cells from T cell lines
13,14
and for isolation of functional antigen-specific, IFN-γ-secreting T cells reacting to other tumor antigens, e.g., SSX
14
, CEA
16
, or HER2
17
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 epitope mapping 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 cells
22
, but not with rhesus macaque cells.
The IFN-γ Secretion Assays can also be used for two-color cytokine analysis
9
and allows counterstaining of peptide-MHC-tetramer-labeled cells
12,15
. It can also be combined with flow cytometric proliferation assays
23
.

Columns

MS, LS, or autoMACS
®
Columns.

References for IFN-γ Secretion Assay – Cell Enrichment and Detection Kit (PE), human

Publications

  1. Brosterhus, H. et al. (1999) Enrichment and detection of live antigen-specific CD4(+) and CD8(+) T cells based on cytokine secretion. Eur. J. Immunol. 29: 4053-4059
  2. Bissinger, A. L. et al. (2002) Isolation and expansion of human cytomegalovirus- specific cytotoxic T lymphocytes using interferon-gamma secretion assay. Exp. Hematol. 30: 1178-1184
  3. Bitmansour, A. D. et al. (2002)
    Direct
    ex vivo
    analysis of human CD4(+) memory T cell activation requirements at the single clonotype level.
    J. Immunol. 169: 1207-1218
  4. Bickham, K. et al. (2001)
    EBNA1-specific CD4
    +
    T cells in healthy carriers of Epstein-Barr virus are primarily Tʜ1 in function.
    J. Clin. Invest. 107: 121-130
  5. Cohen, G. B. et al. (2002) Clonotype tracking of TCR repertoires during chronic virus infections. Virology 304: 474-484
  6. Koehne, G. et al. (2002) Quantitation, selection, and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon-gamma production and growth of cytotoxic precursors. Blood 99: 1730-1740
  7. Altfeld, M. et al. (2001) Vpr is preferentially targeted by CTL during HIV-1 infection. J. Immunol. 167: 2743-2752
  8. Douek, D. C. et al. (2002)
    HIV preferentially infects HIV-specific CD4
    +
    T cells.
    Nature 417: 95-98
  9. Lichterfeld, M. et al. (2004)
    HIV-1-specific cytotoxicity is preferentially mediated by a subset of CD8
    +
    T cells producing both interferon-gamma and tumor necrosis factor-alpha.
    Blood 104: 487-494
  10. Lee, S. K. et al. (2002) The functional CD8 T cell response to HIV becomes type-specific in progressive disease. J. Clin. Invest. 110: 1339-1347
  11. Desombere, I. et al. (2003) The interferon gamma secretion assay: a reliable tool to study interferon gamma production at the single cell level. J. Immunol. Methods 286: 167-185
  12. Meidenbauer, N. et al. (2003) Survival and tumor localization of adoptively transferred Melan-A-specific T cells in melanoma patients. J. Immunol. 170: 2161-2169
  13. Ayyoub, M. et al. (2004)
    An immunodominant SSX-2-derived epitope recognized by CD4
    +
    T cells in association with HLA-DR.
    J. Clin. Invest. 113: 1225-1233
  14. Pittet, M. J. et al. (2001)
    Ex vivo
    IFN-gamma secretion by circulating CD8 T lymphocytes: implications of a novel approach for T cell monitoring in infectious and malignant diseases.
    J. Immunol. 166: 7634-7640
  15. Märten, A. et al. (2002) Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells. Cancer Immunol. Immunother. 51: 25-32
  16. Meyer zu Büschenfelde, C. M. et al. (2001) The generation of both T killer and Tʜ cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells. J. Immunol. 167: 1712-1719
  17. Becker, C. et al. (2001) Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay. Nat Med 7: 1159-1162
  18. Novak, E. J. et al. (2001)
    Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4
    +
    T cell epitopes from complex antigens.
    J. Immunol. 166: 6665-6670
  19. Deniz, G. et al. (2002) Human NK1 and NK2 subsets determined by purification of IFN-gamma-secreting and IFN-gamma-nonsecreting NK cells. Eur. J. Immunol. 32: 879-884
  20. Akdis, M. et al. (2004) Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J. Exp. Med. 199: 1567-1575
  21. Meyer-Olson, D. et al. (2003) Analysis of the TCR beta variable gene repertoire in chimpanzees: identification of functional homologs to human pseudogenes. J. Immunol. 170: 4161-4169
  22. Gutzmer, R. et al. (2003) Human dendritic cells express the IL-18R and are chemoattracted to IL-18. J. Immunol. 171: 6363-6371
  23. Feuchtinger T. et al. (2004)
    Isolation and expansion of human adenovirus-specific CD4
    +
    and CD8
    +
    T cells according to IFN-gamma secretion for adjuvant immunotherapy.
    Exp. Hematol. 32: 282-289

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