For enrichment of HSPCs, BM cells were isolated from femurs of 6- to 7-week-old C57BL/6J mice. Mice were sacrificed and femurs were collected (Kusumbe et al., 2015 (link)). BM cells were isolated by crushing femurs with mortar and pestle in Ca2+/Mg2+-free PBS containing 2% heat-inactivated bovine serum. The cells were drawn by passing through a 25G needle several times and filtered with a 70-μm filter. Single-cell suspension obtained was subjected to lineage depletion (MACS, Miltenyi Biotech) following the manufacturer’s instructions.
Lineage-depleted BM cells were labeled with cell membrane dyes PKH67 green fluorescent linker kit (Sigma). 5 × 106 cells were washed once in DMEM and suspended in 1 mL of diluent solution C. 1 mL of PKH67 at 2 × 10−3 M in diluent C was added and mixed, and cells were incubated for 7 min at room temperature (RT). The dye was inactivated by adding 1 mL DMEM supplemented with 25% fetal calf serum. This mixture was centrifuged, and cells were washed twice and suspended in sterile ice-cold PBS.
Lineage-depleted BM cells were labeled with cell membrane dyes PKH67 green fluorescent linker kit (Sigma). 5 × 106 cells were washed once in DMEM and suspended in 1 mL of diluent solution C. 1 mL of PKH67 at 2 × 10−3 M in diluent C was added and mixed, and cells were incubated for 7 min at room temperature (RT). The dye was inactivated by adding 1 mL DMEM supplemented with 25% fetal calf serum. This mixture was centrifuged, and cells were washed twice and suspended in sterile ice-cold PBS.