Cd150

Bone marrow cells were flushed from the long bones with Dulbecco’s Modified Eagle’s Medium supplemented with 5% heat-inactivated calf serum (Gibco). To obtain a single cell suspension, cells were filtered through a 45 µm nylon screen. Cells were blocked with anti-rat and anti-mouse IgG (Sigma) for 15 min. For isolation of HSCs, cells were then stained with CD45 (APC-Cy7, BD Pharmingen), CD150 (APC, Biolegend), CD48 (Pacific Blue, Biolegend), SCA1 (PE-Cy5.5, Invitrogen), and c-KIT (PE-Cy7, eBioscience), as well as PE-conjugated lineage markers including CD19, B220, CD3, CD4, CD8, GR-1, MAC-1, NK1.1, and TER119 (eBioscience). Cells were resuspended in DAPI to discriminate live from dead cells. HSCs were sorted by isolating Lineage^neg, SCA1⁺, c-KIT⁺, CD150⁺, CD48^neg cells using a BD FACSAria II flow cytometer (BD Biosciences). Approximately 150,000–2,000,000 CD45⁺ cells or 2300–4400 HSCs, or ~150,000–2,000,000 CD45^neg cells were resuspended in phosphate buffered saline with 100,000–200,000 whole bone marrow cells for reconstitution and injected into lethally irradiated isogenic wild type host mice. For Sm22-Cre;R26R BMT experiments, cells were transplanted into immunodeficient Rag1^−/− mice. Donor chimerism was assessed by measuring GFP or CD45.1 vs. CD45.2 levels by FACS in the peripheral blood 8 weeks after transplantation.

The peripheral blood cells and splenocyte cells from various treatments were stained with anti- CD16/32, CD3, CD4, CD8, and CD19 antibodies. For thymocytic cells CD117, CD44, CD25, CD127, CD3, CD4 and CD8 were used to stain different population. Hematopoietic stem and progenitor cells were stained with lineage markers as described earlier [18 (link)]. We used following antibodies: CD117, Ly-6A/E, CD34, CD135, CD127, CD48, CD150 and CD16/32 from BD biosciences and Mac-1, Gr-1, CD4, CD8, B220, CD3, and Ter119 from Biolegend. Cells were analyzed by MACSQuant Analyzer flow cytometer (Miltenyi Biotec) and data analyzed with FlowJo software (Tree Star Inc.).