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

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

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.

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

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.

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

invitro^1,2,3,5,6,9

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.

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

Documents and Protocols

App notes/customer reports

Effector memory T helper cells secrete IFN-γ upon stimulation with cytokines: a role in chronic inflammation.

Application protocol

Expansion of antigen-specific cytokine-secreting T cells

Protocols

Detection and enrichment of cytokine-secreting cells from whole blood

Protocols

Detection and enrichment of cytokine-secreting cells

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

Publications

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
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
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
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
Cohen, G. B. et al. (2002) Clonotype tracking of TCR repertoires during chronic virus infections. Virology 304: 474-484
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
Altfeld, M. et al. (2001) Vpr is preferentially targeted by CTL during HIV-1 infection. J Immunol 167: 2743-2752
Douek, D. C. et al. (2002)
HIV preferentially infects HIV-specific CD4
⁺
T cells.
Nature 417: 95-98
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
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
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
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
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
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
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
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
Becker, C. et al. (2001) Adoptive tumor therapy with T lymphocytes enriched through an IFN-gamma capture assay. Nat Med 7: 1159-1162
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
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
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
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
Gutzmer, R. et al. (2003) Human dendritic cells express the IL-18R and are chemoattracted to IL-18. J Immunol 171: 6363-6371
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

Data and images