Ter119 fitc

To evaluate donor chimerism, PB samples were treated with a red blood cell (RBC) lysis buffer, and stained with CD45.2 (Ly5.2)-Cy5 (eBioscience) for S-HSCT or Ly9.1-FITC (BD Pharmingen) for A-HSCT, as follows. The cells were resuspended with Hank’s Balanced Salt Solution (HBSS) (Ca²⁺, Mg²⁺ free, Invitrogen, CA) containing 2% fetal bovine serum (FBS) (HF2 solution) and then incubated for 30 min on ice with fluorochrome-conjugated antibodies. Following staining, cells were washed twice with phosphate buffered saline (PBS) and then resuspended with HF2 containing 1ug/ml of propidium iodide (Sigma). The analyses were performed using a dual laser FACScan (BD Bioscience). Mice that were clinically ill were euthanized using CO₂ narcosis. BM, spleen, and thymus were harvested and stained with antibodies to Mac-1-PE (BD Pharmingen), Gr-1-FITC (BD Pharmingen), B220-APC (BD Pharmingen), CD4-PE (eBioscience), CD8-FITC (eBioscience), CD71-PE (eBioscience), Ter119-FITC (BD Pharmingen), cKit-FITC (BD Pharmingen), Sca-1-PE (BD Pharmingen), and analyzed as described above. Cell quest pro version 5.2.1 (BD Bioscience) software was used for flow cytometry analysis.

For mouse phenotyping peripheral blood and bone marrow from femurs were collected from animals of different genotypes and processed as previously described.^{16 (link)} For flow analysis, samples were stained with Gr1-PE (Miltenyi Biotec, Woking, UK), CD11b (Mac1)-FITC (Becton Dickinson), CD71-PE (Becton Dickinson), Ter119-Fitc (Becton Dickinson). For analysis of progenitor populations BM cells were lysed with Red Blood Cell buffer (NH4, Cl) and then enriched with the EasySep Hematopoietic Progenitor Cell Isolation Kit (StemCell Technologies, Cambridge, UK) followed by staining with following antibodies Sca1-PB, c-KIT-Acy7, CD34-FITC, Flk2-PeCy5, Cd16/32-PE, CD150-PeCy7, CD48-APC.