Thymocytes were isolated from 4–6-wk-old Bcl11b-YFP reporter mice. Lin+ cells were depleted by staining with biotinylated antibodies against CD8α (eBioscience; 13–0081-86), TCRγδ (13–5711-85), TCRβ (13–5961-85), TER-119, NK1.1, Dx5 (13–5971-82), CD11c, and CD11b, then incubating cells with streptavidin magnetic beads, followed by passing them through an LS magnetic column (Miltenyi Biotec) in accordance with the manufacturer’s instructions. Purified DN cells were stained with c-Kit APCe780 (eBioscience; 17–1171-82), CD25 PE (12–0251-28), CD44 e450 (48–0441-82), and streptavidin PerCPCy5.5 (45–4317-80) and then subjected to intracellular staining for GATA3 AF647 (BD PharMingen; 560068) or PU.1 AF647 (Cell Signaling; 2240) using the BrdU Flow Kit (BD PharMingen).
For staining of sgRNA-introduced BM cells, surface antibodies against CD45 PECy7 (eBioscience; 25–0451-82), c-Kit APC, CD25 APC-e780 (47–0251-82), human-NGFR PE (12–9400-42), and a biotin-conjugated lineage cocktail (CD8α, CD11b, CD11c, Gr-1, TER-119, NK1.1, CD19, TCRβ, and TCRγδ) with streptavidin PerCPCy5.5 were used for staining.
Prior to cell surface staining, cells were treated with 2.4G2 cell supernatant. All of the cells were analyzed using flow cytometers, MacsQuant 10 (Miltenyi Biotec), LSRFortessa (BD PharMingen), or FACSAria (BD PharMingen) with FlowJo software (Tree Star).
For staining of sgRNA-introduced BM cells, surface antibodies against CD45 PECy7 (eBioscience; 25–0451-82), c-Kit APC, CD25 APC-e780 (47–0251-82), human-NGFR PE (12–9400-42), and a biotin-conjugated lineage cocktail (CD8α, CD11b, CD11c, Gr-1, TER-119, NK1.1, CD19, TCRβ, and TCRγδ) with streptavidin PerCPCy5.5 were used for staining.
Prior to cell surface staining, cells were treated with 2.4G2 cell supernatant. All of the cells were analyzed using flow cytometers, MacsQuant 10 (Miltenyi Biotec), LSRFortessa (BD PharMingen), or FACSAria (BD PharMingen) with FlowJo software (Tree Star).