Lineage depletion kit

The femurs, tibiae, iliac crest, humeri, and vertebrae of 6-16 weeks old mice were crushed, washed with 10 mL of PBS supplemented with 2% heat-inactivated FBS, and strained through 70-μm meshes. Cell suspension was depleted of red blood cells by ammonium chloride lysis (STEMCELL Technologies), and stained with the lineage depletion kit (19816A, STEMCELL Technologies) following the manufacturer’s instructions and passed through magnetic columns. Lineage-depleted cells were resuspended in 100 μl of PBS supplemented with 2% FCS containing the following antibodies against: c-Kit (APC-Cy7, clone 2B8, 105826, Biolegend), Sca-1 (BV421, clone D7, 108128, Biolegend), CD45 (FITC, clone 30-F11, 103108, Biolegend), Flt3 (PE, clone A2F10, 135306, Biolegend) and Il-7R⍺ (BV605, clone A7R34, 135041, Biolegend). Cells were incubated at 4°C for 30 minutes in the dark, washed, and resuspended in 100 μl of PBS supplemented with 2% FCS containing streptavidin (BV510, 405234, Biolegend). Cells were further incubated at 4°C for 15 minutes, washed and resuspended in 500 μl of PBS supplemented with 2% FCS containing 0.5 μl 7AAD (A1310, Life Technologies). Cells (lineage^- c-Kit⁺ population, and lineage^- c-Kit^lo Sca-1⁺ population) were bulk sorted using a Becton Dickinson Influx sorter.