OP9 bone marrow stromal cells expressing the Notch ligand DL-1 (OP9-DL1), provided kindly by Dr J.C. Zúñiga-Pflücker (University of Toronto, Toronto, Canada), were cultured and maintained in α-MEM (Gibco) medium with 10% fetal bovine serum (Gibco). For DN3 cells co-cultures, the sorted DN3 cells were plated onto confluent OP9-DL1 monolayers (70–80% confluent) and cultured with of 5 ng ml−1 recombinant murine interleukin-7 (Peprotech) and 5 ng ml−1 Flt3L (Peprotech).
Retrovirus preparation was performed in Plat-E cells. Plat-E cells were transfected with pMX-IRES-GFP containing indicated genes, the medium were replaced with fresh medium after 10 h, and retrovirus supernatant was collected after additional 72 h.
Expression of ectopic proteins in DN3 cells was performed as previously described49 (link). In brief, RetroNectin (Takara) coating and washing were carried out according to the manufacturer's instruction. The retrovirus was added to wells coated with RetroNectin, followed by 4 h incubation at room temperature and removal of the retrovirus. After 24 h co-culture, DN3 cells were directly placed on plates coated with RetroNectin and retrovirus. And trypsinized OP9-DL1 cells were added to the DN3 cells.
Retrovirus preparation was performed in Plat-E cells. Plat-E cells were transfected with pMX-IRES-GFP containing indicated genes, the medium were replaced with fresh medium after 10 h, and retrovirus supernatant was collected after additional 72 h.
Expression of ectopic proteins in DN3 cells was performed as previously described49 (link). In brief, RetroNectin (Takara) coating and washing were carried out according to the manufacturer's instruction. The retrovirus was added to wells coated with RetroNectin, followed by 4 h incubation at room temperature and removal of the retrovirus. After 24 h co-culture, DN3 cells were directly placed on plates coated with RetroNectin and retrovirus. And trypsinized OP9-DL1 cells were added to the DN3 cells.