Isolation and Culture of Mouse LSK Cells

Using a FACSAria II cell sorter (BD), LSK cells were separated based on their high expression of SCA-1 and c-KIT from lineage-depleted bone marrow, as described in Dallas et al. (2005) (link). Independent LSK isolates were cultured on immobilized Delta1 (Varnum-Finney et al., 2000 (link)), as previously described (Varnum-Finney et al., 2003 (link)). In brief, non-tissue culture-treated flasks (Nunc) were incubated overnight at 4°C with Delta1^ext-IgG (0.75 or 5 μg/mL) or human IgG₁ (5 μg/mL, Sigma I4506) diluted in PBS, together with 5 μg/mL Retronectin (Takara). Flasks were washed extensively with PBS. Isolated LSK cells were added to prepared flasks in Iscove’s modified Dulbecco’s medium supplemented with 20% fetal bovine serum and 4GF (100 ng/mL murine SCF, human FLT-3 ligand, and human IL-6; 10 ng/mL human IL-11). Each Delta1^ext-IgG culture was independently treated with IL-7 (100 ng/mL) at day 14 to drive cells further into T cell development. From each biological LSK replicate, DN1 (KIT⁺⁺CD44⁺CD25⁻) cells were isolated from 4GF conditions, whereas DN2a (KIT⁺⁺CD44⁺CD25⁺) and DN2b (KIT⁺CD44^loCD25⁺) subpopulations were isolated from 4GF + IL-7 conditions using anti-mouse monoclonal antibodies CD25-APC-Cy7 (clone PC61, BD catalog no. 557658), CD44-APC (clone IM7, BD catalog no. 559250), and c-KIT-PE-Cy5 (clone 2B8, eBioscience catalog no. 15-1171-82).