Isolation and Analysis of Hematopoietic Stem Cells

For flow cytometry analysis and isolation of hematopoietic stem and progenitor cells, cells were stained with relevant antibodies^{51 (link)} in PBS with 2% fetal bovine serum for 30−45 min on ice. Antibody clones that were used: Sca-1-PE-Cy7, c-Kit-APC, CD150-PE, CD48-BV421, CD45.1-FITC, CD45.2 PE-Cy5, Gr-1-PE-Cy5, Mac1-PE-Cy5, IgM-PE-Cy5, CD3-PE-Cy5, CD4- PE-Cy5, CD8-PE-Cy5, CD45R-PE-Cy5 and Ter-119-PE-Cy5. Detailed antibody information is summarized in Supplementary Table S6. HSPCs were stained with a lineage antibody cocktail (Gr-1, Mac1, CD3, CD4, CD8, CD45R, TER119 and B220), Sca-1, c-Kit, CD150 and CD48. Cell types were defined as followed: LSK compartment (Lin⁻Sca-1⁺c-Kit⁺), LT-HSC (LSK CD150⁺CD48⁻), ST-HSC (LSK CD150⁻CD48⁻), MPP2 (LSK CD150⁺CD48⁺) and MPP3/4 (LSK CD150⁻CD48⁺). B cells (CD45.2⁺Mac1⁻Gr-1⁺B220⁺), T cells (CD45.2⁺Mac1⁻Gr-1⁺CD3⁺) and myeloid cells (CD45.2⁺Mac1⁺Gr-1⁻). Samples were analyzed on a flow cytometer (CytoFLEX LX, Beckman). For sorting HSCs, lineage antibody cocktail-conjugated paramagnetic microbeads and MACS separation columns (Miltenyi Biotec) were used to enrich Lin⁻ cells before sorting. Stained cells were re-suspended in PBS with 2% FBS and sorted directly using the Beckman moflo Astrios EQ (Beckman). Flow cytometry data were analyzed by FlowJo (BD) software.