Fig 1: Schematic diagram of generating functional T lymphocytes by reprogramming in vivo. The recombinant Hoxb5 vector or GFP empty vector control was transduced into Plat-E cells to produce viruses. Pro/pre-B cells (Ter119–Mac1–CD3–CD4–CD8–B220+CD19+CD93+IgM–) were sorted from the bone marrow and then cultured in medium (15%FBS, 100 mM GlutaMAX, 10-4 M ß-ME, 10 ng/ml mSCF, 10 ng/ml Flt3L and 10 ng/ml IL7) for 12–16 h before virus transduction. Pro/pre-B cells (1 × 106 per ml) were transduced with the mixed viruses with equal pre-adjusted titers. The pro/pre-B cells transduced with retro-Hoxb5 virus were collected for transfection efficiency analysis and then were transplanted into sublethally irradiated syngeneic recipient mice via orbital veins. Four weeks after transplantation, cells harvested from PB, spleen, lymph node and thymus were analyzed by flow cytometry.
Fig 2: Flow cytometry analysis of the purity of sorted Ter119–Mac1–CD3–CD4–CD8–B220+CD19+CD93+IgM–pro/pre-B cells. Bone marrow nucleated cells were first incubated with biotin-conjugated anti-B220 antibody and then enriched by streptavidin microbeads using AutoMACS Pro. The pro/pre-B cells were further sorted from the B220 enriched cells by defined markers. Flow cytometry analysis confirmed the purities of B220 enriched cells (top) and sorted pro/pre-B cells (bottom).
Fig 3: Flow cytometry analysis of induced T lymphocytes in irradiated recipients injected with Hoxb5-retrovirus transduced pro/pre-B cells. GFP+Ter119–Mac1– populations obtained from the PB, spleen, and LNs (a) or GFP+Ter119–Mac1–CD19– iDN populations from the thymus (b) of recipient mice were analyzed. Three million pro/pre-B cells transduced with the retro-Hoxb5 virus were transplanted into sublethally irradiated syngeneic mice. Four weeks after transplantation, the single nucleated blood cells harvested from PB, spleen, lymph node and thymus were analyzed by flow cytometry. Representative plots were shown.
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