Isolation and Sorting of Hematopoietic Stem Cells

Cell sorting protocols were as previously described [28 (link), 32 (link)]. Briefly, bone marrow was flushed in PBS supplemented with 0.1% bovine serum albumin and 2 mM EDTA, filtered through 40 μm cell strainers, and subjected to red blood cell lysis in ACK buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA). The samples were stained with biotin anti-mouse Lineage Panel (BioLegend), PE-Cy7 cKit (clone 2B8, BioLegend), APC-Cy7 Sca-1 (D7, BioLegend), FITC CD48 (HM48-1, eBioscience), APC CD150 (TC15-12F12.2, BioLegend), PE Flt3 (A2F10, eBioscience), Brilliant Violet 421 CD34 (RAM34, BD Biosciences), and streptavidin-PECy5 (BioLegend). Cell sorting was performed on FACSAria, with the FACS Diva software (BD Biosciences).