YILIMART - Rapid Cytokine Inspektor (CD4 T Cell), h

T lymphocytes execute and control immunological reactions with a repertoire of cytokines, cytotoxic substances, and other mediators. The quantitative and qualitative analysis of CD4

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T cells specifically recognizing and reacting towards a defined antigen provide important information to understand their function in various immunological situations. Antigen-specific CD4

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T cells can be identified and characterized by analyzing their effector function, e.g., production of cytokines and expression of CD154.

The Rapid Cytokine Inspector (CD4 T Cell) Kit, human, has been developed for the fast and easy evaluation of cytokine expression in activated CD154

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CD4

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T cells by intracellular staining. The kit contains an antibody cocktail for the identification of CD154

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CD4

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T cells and the exclusion of monocytes and B cells as well as brefeldin A and reagents for the fixation and permeabilization of cells after T cell stimulation. The kit is optimized for use in combination with Rapid Cytokine Inspector Anti-Cytokine antibodies of interest (not included in the kit).

Detailed procedure

PBMCs are cultured with or without antigen for a total of 6 hours. Upon activation, the CD4

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T cells start to produce cytokines and upregulate CD154 expression. After 2 hours of cultivation, brefeldin A is added to the cells to inhibit transport of proteins to the cellular membrane. After stimulation, the culture supernatant is removed, and the CD4 T Cell Detection Cocktail is added together with Rapid Cytokine Inspector Anti-Cytokine antibodies of interest. Cell surface markers are stained during a short incubation step. Fixation and permeabilization of the cells is performed and enables subsequent intracellular staining of cytokines and CD154. The cells are washed and analyzed by flow cytometry. The use of 96-well cell culture plates enables the analysis on multiple samples in parallel. Washing steps after T cell stimulation, and before immunofluorescent staining, fixation, and permeabilization are not required.

Applications

● High-throughput multiparameter analysis of antigen-specific T cells.

● Rapid identification and enumeration of cytokine-producing activated antigen-specific CD154

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CD4

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T cells upon

in vitro

stimulation with the respective antigen or upon polyclonal restimulation.

● Immunomonitoring of antigen-specific T cells.

Figure 1

Human PBMCs were incubated with or without PepTivator CMV pp65 for 6 hours; brefeldin A was added after 2 hours. The cells were stained with the CD4 T Cell Detection Cocktail and Rapid Cytokine Inspector Anti-IFN-γ-PE and analyzed by flow cytometry using the MACSQuant® Analyzer. IFN-γ and CD154 expression was analyzed for CD4

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T cells in stimulated and unstimulated samples*.

A:	B:
Stimulated sample	Unstimulated control

*Please refer to the data sheet for detailed gating strategy and analysis.

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Rapid Cytokine Inspektor (CD4 T Cell), h

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Detailed procedure

Applications

Figure 1

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