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iTAg MHC Tetramer IFN-γ Kit. Enhanced Cellular Analysis.
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For the first time, Tetramer analysis combined with intracellular cytokine staining (ICC) provides both function and antigen specificity. This synergistic approach has been developed and optimized to clearly separate CD8+ T cells into distinct sub-populations, enabling simultaneous analysis on both the quantitative and functional parameters.
- Measures functional status of immune response targeted to specific allele(s) and antigens
- Analyze quantitative and functional parameters simultaneously using a single sample
- Stratify specific and non-specific cytokine generation, providing a more accurate immune response assessment
- Eliminate possible overestimation of total immune response to a particular antigen that can occur with ICC staining alone
The ICC IFN-γ Kit. What's included.*
Each individual kit performs 50 tests using recommended panel and includes:
- Anti-IFN-γ FITC
- Lyse Reagent
- Fixative Reagent
- Permeabilization Reagent
- Brefeldin A (for use with iTAg MHC Tetramers from Beckman Coulter. Appropriate for use with all Class I alleles)
A) Functional IFN-γ analysis
Measurement of cytokine producing cells using the iTAg MHC Tetramer IFN-γ Kit without the addition of Tetramer (Figure A) is comparable to the total IFN-γ + cells using the iTAg MHC Tetramer IFN-γ+ Kit with Tetramer (Figure E). Total IFN-γ measurement using this assay corresponds to traditional ICC analysis.
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B) iTAg MHC Tetramer analysis
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C)
Detection and enumeration of an antigen-specific T cell population using an iTAg MHC Tetramer assay with conventional staining (Figures B and C) is comparable to the total Tetramer + population using the iTAg MHC Tetramer IFN-γ Kit (Figures D and E), demonstrating recovery of the Tetramer + T cells after peptide stimulation, incubation and permeabilization.
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D) iTAg MHC Tetramer IFN-γ Assay
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E*
Enhanced T cell analysis is provided by separating CD8 + T cells into distinct sub-populations based on parameters of iTAg MHC Tetramer positivity and IFN-γ positivity. This differentiates antigen-specific, IFN-γ + cells (Figure E, Quadrant E2) from non-specific, IFN-γ + (Quadrant E4). Traditional ICC methods may include the measurement of a non-specific, IFN-γ producing T cell population. This population varies widely and is both donor and epitope dependent.
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*In this example 36% of the total antigen-specific population is IFN-γ+
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iMASC™ Antibody Gating Kit
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The iMASC Antibody Gating Kit increases the sensitivity and improves signal to noise ratio of iTAg MHC Tetramer Analysis. The Kit, used with SA-PE labeled iTAg Tetramers, will enrich for CD8* events in multi-parameter flow cytometric data-space without cell sorting. This approach effectively excludes platelets and debris by morphology, and captures blasting cells, which are known to be donor and/or sample processing dependent. While capturing rare antigen specific T cells of interest, the iMASC strategy removes potential background fluorescence caused by non-viable cells and/or Tetramers, and increases the detection levels, facilitating statistically significant acquisitions.
The iMASC Antibody Gating Kit includes the following:
- anti-CD8/FITC
- anti-CD4/PC5
- anti-CD13/PC5
- anti-CD19/PC5
Example Histograms/Gating
The information below illustrates the iMASC gating strategy, which utilizes a sequential gating approach and dual parametric histograms to resolve the antigen specific T cell population.
Dot plot 1
Dot plot 1 represents the iMASC gate (A) which excludes CD4, CD13 and CD19 from the analysis.
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Dot plot 2
Dot plot 2 is generated from region A. Region B is drawn, based on morphology, and excludes debris from the gate.
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Dot plot 3
Dot plot 3 is generated from regions A and B. Region C is drawn to include CD8+ T cells only.
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Dot plot 4
Dot plot 4 is generated from regions A, B and C. Region D represents antigen specific CD8+/iTAg Tetramer positive T cells.
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iTAg™ MHC Tetramer Lyse and Fixative
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iTAg MHC Tetramer Lyse and Fixative are optimize for use with Immunomics branded products. It is recommended for use with whole blood and with all iTAg MHC Tetramers to preserve the integrity of the cells:
- Clear definition of lymphocyte, monocyte and granulocyte populations
- Less debris in sample populations
- For whole blood, iTAg Lyse is most effective when used with iTAg Fixative
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