Recombinant mouse il 7

For in vivo limiting dilution assay, we transplanted limiting numbers of total BM cells (1 × 10⁴, 4 × 10⁴, 8 × 10⁴, and 2 × 10⁵) isolated from primary recipients (control and Pcgf1^Δ/Δ mice 1 mo after tamoxifen injections) with CD45.1⁺ competitor total BM cells (2 × 10⁵) into lethally irradiated CD45.1 recipient mice. PB analyses were performed at 16 wk after transplantation.
For in vitro limiting dilution assay, we sorted HSCs from control and Pcgf1^Δ/Δ mice 1 mo after tamoxifen injections and cultured limiting numbers of the cells (1, 5, 25, and 125) with TSt-4 (B cells) or TSt-4/DLL1 stromal cells (T cells) in RPMI (Thermo Fisher Scientific) supplemented with 10% BSA (093001; STEMCELL Technologies), 50 μM 2-ME (Sigma-Aldrich), 100 μM MEM Non-Essential Amino Acids solution (Gibco), 100 μM sodium pyruvate (Gibco), and 2 ng/mL recombinant mouse IL-7 (577802; BioLegend) for 28 d. The generation of CD19⁺ B cells or Thy1.2⁺ T cells in each well was detected by flow cytometry.

OP9-DL1 cells, kindly provided by Maria Ciofani (Duke University), were cultured in MEMα (Gibco) supplemented with 10% FBS (Atlanta Biologicals) and 1× penicillin/ streptomycin (Gibco). HEK293T cells were cultured in DMEM supplemented with 10% FBS, 1× penicillin/ streptomycin, 1× non-essential amino acids, and 1× GlutaMAX. For OP9-DL1 culture of thymocytes, cultures were additionally supplemented with 5 ng/mL recombinant mouse IL-7 (Biolegend). Scid.adh.2c2 cells were cultured in IMDM supplemented with 10% FBS (Hyclone), 1× penicillin/ streptomycin, 1× NEAA, 1× sodium pyruvate, 1× GlutaMAX, and 55 µM β-mercaptoethanol. In some OP9-DL1 cultures 0, 1, 10, 100, or 1000 nM rapamycin (Cayman Chemicals), 0, 1, 10, 100, or 1000 nM everolimus (Cayman Chemicals), 10 µg/mL anti-mouse IFNAR-1 antibody (Biolegend), 5 µg/mL RU.521 (Invivogen), 0.5 µg/mL H-151 (Cayman Chemicals) or 25 or 250 µg/mL Cridanimod (Cayman Chemicals) were added. All cultures were maintained at 37 °C with 5% CO₂.