Cd117 apccy7

BM from one femur was flushed with PBS, made into single cell suspension with a syringe, and centrifuged. Pellet with then treated with RBC lysis solution for 5 min. After re-suspension in PBS and washing cells were stained with lineage markers on APC (CD4, CD5, CD8, CD11b, B220 (CD45R), Gr1, Ter119, CD41) (eBioscience), CD117 APCCy7 (eBioscience), Sca 1 PECy7 (eBioscience), CD150 PE (Biolegend), Endoglin Pacific Blue (Biolegend), CD16/32 PerCP5.5 (eBioscience) and gating as described by Pronk et al. [41] (link). Initial gating identified lineage (CD4, CD5, CD8, CD11b, B220, Gr1, Ter119, CD41) negative singlet cells. Cells were classified as Lin⁻ Sca⁺ Kit⁺ (LSK) or Lin⁻ Kit⁺ (LK). LSK cells were further subdivided between Endoglin⁻ CD150⁻ multipotent progenitors (MPP) or Endoglin⁺ CD150⁺ long term hematopoietic stem cells (LT-HSC). LK cells were further subdivided between CD16/32⁺ CD150⁻ granulocyte macrophage progenitors (GMP) or CD16/32⁻ CD150⁻Endoglin⁻ pre-granulocyte macrophage (Pre-GM) cells. Representative sample showed in Supplementary Figure 1B. In competitive BMTs CD45.1 and CD45.2 stains were used for determining donors in the blood and SVF. Within the BM compartment and HSC staining CD45.2 staining was used to differentiate the CD45.2 and CD45.1 donor groups.