YILIMART - Rapid Cytokine Inspektor (CD4/CD8 TC), 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

⁺

T cells and CD8

⁺

T cells specifically recognizing and reacting towards a defined antigen provide important information to understand their function in various immunological situations. Antigen-specific T cells can be identified and characterized by analyzing their effector function, e.g., production of cytokines.

The Rapid Cytokine Inspector (CD4/CD8 T Cell) Kit, human, has been developed for the fast and easy evaluation of cytokine expression in activated T cells by intracellular staining. The kit contains an antibody cocktail for the identification of CD4

⁺

and CD8

⁺

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 cells start to secrete cytokines. 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/CD8 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. The cells are washed and analyzed by flow cytometry. 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 CD4

⁺

and CD8

⁺

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/CD8 T Cell Detection Cocktail and Rapid Cytokine Inspector Anti-IFN-γ-PE. IFN-γ expression was analyzed for CD4

⁺

and CD8

⁺

T cells in stimulated and unstimulated samples*.

A:	B:
Stimulated sample	Unstimulated control

C:	D:
Stimulated sample	Unstimulated control

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

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Rapid Cytokine Inspektor (CD4/CD8 TC), h

For research use only

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Detailed procedure

Applications

Figure 1

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