Mac1 pe

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.

Flow cytometry was performed on bone marrow, spleen, and thymus samples. For the latter two, cells were forced through a 40 µm nylon cell strainer in PBS with 2% fetal calf serum (FCS). For the bone marrows, cells were collected by flushing femurs and tibias with PBS containing 2% FCS. After RBC lysis, collected cells were washed and resuspended in PBS with 2% FCS and counted on a hemocytometer. For fluorescence-activated cell sorting (FACS) analyses, cells were stained for 30 mins at 4 °C with the following antibodies: CD3 FITC (BioLegend San Diego, CA; 17A2), CD4 PE (BD Biosciences; GK1.5), CD8 APC (BD Biosciences; 53–6.7), CD19 BV605 (Biolegend; 6D5), CD43 APC (Biolegend; S11), IgD e450 (eBiosciences; 11–26 Waltham, MA), IgM PE-Cy-7 (Biolegend; RMM-1), B220 PE (eBiosciences RA3–6B2), Gr-1 FITC (Biolegend RB-6–8C5), Mac1 PE (BD Biosciences; M1/70), and CD11c APC (eBiosciences; N418). FACS analysis was performed on a LSR Fortessa flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences). Data was analyzed with FlowJo software (Tree Star Inc, Ashland, OR).