Computational protocol: GATA-3 is required for early T lineage progenitor development

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Protocol publication

[…] Single-cell suspensions of thymocytes, bone marrow, fetal liver, or fetal spleen cells were first incubated with FcBlock (BD) and then incubated with various combinations of antibodies (BD or eBioscience). The following antibodies were used: CD4 (RM4-5), CD8 (53-6.7), c-Kit (2B8), CD44 (IM7), CD25 (PC61), CD45.2 (104), CD45.1 (A20), CD3 (145-2C11), TCRβ (H57-597), γδTCR (GL3), NK1.1 (PK136), CD11C (N418), B220 (RA3-6B2), CD19 (1D3), Mac-1 (M1/70), Gr-1 (RB6-8C5), TER119 (TER119), IL-7Rα (A7R34), PIR-A/B (6C1), Sca1 (D7), Flt3 (A2F10), CD93 (AA4.1), Ly-6c (AL-21), CD49b (DX5), and CD27 (LG.7F9). Immature T cells were analyzed as previously described (). The following cocktail of antibodies was used to exclude mature hematopoietic lineage cells (Lin+) in the thymus and the cells cultured in the OP9-DL co-culture system: CD8, CD3, TCRβ, γδTCR, NK1.1, CD11c, Mac-1, Gr-1, B220, CD19, and Ter119. In some experiments, CD8+ cells were removed before flow cytometric analysis. T-restricted progenitors in the fetal liver were analyzed as previously described (). A cocktail containing antibodies recognizing TER119, Gr-1, B220, CD19, NK1.1, and Thy1.2 was used to exclude Lin+ cells in the fetal liver. Progenitors in the bone marrow were analyzed as previously described (; ). A cocktail containing antibodies recognizing TER119, Gr-1, B220, CD19, CD3, and Mac-1 was used to exclude Lin+ cells in the bone marrow for the analysis of HSCs, MPPs, and LMPPs. A cocktail containing antibodies recognizing TER119, Mac-1, CD19, CD3, NK1.1, CD49b, Ly-6c, and Gr1 was used to exclude Lin+ cells in the bone marrow for the analysis of CLP and Fr.A. Stained cells were analyzed or sorted using flow cytometers (FACSCanto II or FACSAria; BD). Doublets were excluded with forward-scatter area versus width pulses. Dead cells were excluded by analyzing DAPI or propidium iodide (PI). The resulting cell distribution files were analyzed using FACSDiva (BD), Weasel (WEHI Biotechnology Centre), or FlowJo (Tree Star, Inc.) software. […]

Pipeline specifications

Software tools Weasel, FlowJo
Application Flow cytometry
Organisms Mus musculus