MBTP 33: Immunophenotyping of activated T cells within antigen-stimulated human PBMCs using flow cytometry

Miltenyi Biotec-tested panel 33 (MBTP 33)

This application protocol describes the characterization of activated T cells within antigen-stimulated peripheral blood mononuclear cells (PBMCs) via simultaneous measurement of surface markers and intracellular cytokines using flow cytometry. Prior to antibody staining PBMCs were stimulated with a cocktail of PepTivator CMV pp65, PepTivator EBV consensus, and PepTivator AdV5 Hexon from Miltenyi Biotec.

Protocol

Figure 1: Gating strategy showing the analysis of T cell subsets within antigen-stimulated human PBMCs. Samples were initially gated on lymphocytes based on SSC-A/FSC-A gating (A). Single cells were gated using FSC-H/FSC-A and SSC-A/SSC-H gating (B, C). Dead cells and other unwanted cells were excluded (D). T cells were gated as CD3+ cells (E). Subsets of T cells were further gated based on CD4 and CD8 expression (F). 

Figure 2: Gating strategy showing the analysis of the activation phenotype of different T cell subsets. Samples were initially gated as shown in Figure 1. The activation phenotype of the different T cell subsets CD3+ cells (A, B), CD4+ cells (C, D), CD8+ cells (E, F), and CD4 CD8 cells (G, H) was characterized based on the level of expression of the intracellular cytokines TNF-α and IFN-γ and of the surface marker CD154.

Materials

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