Intracellular Staining by Flow Cytometry
Detection of T‑bet/TBX21 in Human PBMCs Stimulated to Induce Th1 Cells by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) either (A) untreated or (B) treated with Anti-Human IL-4 Polyclonal Antibody (Catalog # AB-204-NA; 20 μg/mL) and 10 ng/mL Recombinant Human IL‑12 (Catalog #|
219-IL) for 5 days to induce Th1 cell development were stained with Mouse Anti-Human T‑bet/TBX21 Monoclonal Antibody (Catalog # MAB53851) followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B) and Mouse Anti-Human CD4 APC‑conjugated Monoclonal Antibody (Catalog # FAB3791A). Quadrant markers were set based on control antibody staining (Catalog # MAB002). To facilitate intracellular staining, cells were fixed and permeabilized with FlowX FoxP3 Fixation & Permeabilization Buffer Kit (Catalog # FC012).
T-box expressed in T cells (T-bet), also known as T-box transcription factor TBX21, is a 62 kDa member of the T-box family of transcription factors and the Tbr1 subfamily. Human T-bet is 535 amino acids in length and contains a T-box DNA binding domain (aa 136-327). Human T-bet shares 88% aa sequence identity with mouse T-bet. T-bet is a nuclear protein highly apparent in Th1 cells. Northern blot analysis revealed that it is also expressed in lung, thymus and spleen. Functionally, T-bet controls the expression of the Th1 cytokine, IFN-gamma, and initiates Th1 lineage development from naïve Th precursor cells by both activating Th1 genetic programs and by repressing the opposing Th2 programs.
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