Anti human cd45 apc antibody clone hi30

NSG adult female mice (6–8 weeks old) were purchased from Charles River and were sub-lethally irradiated (3 Gy) 24 h prior to transplantation. CD34+ HSPCs were thawed and cultured. At day 4 of culture (2 days after editing), 0.5 × 10⁶ viable cells/mouse were injected via tail vein into NSG mice with a 27-gauge × 0.5-inch needle. The same cell number per mouse was used for all experimental conditions tested. After 8 and 14 weeks post-transplantation, mice PB from the tail vein was lysed with 1× RBC lysis buffer (ThermoFisher Scientific) and stained with anti-human CD45 APC antibody (clone HI30, BioLegend) to evaluate human CD45+ engraftment by flow cytometry. At 14–16 weeks post-transplantation, level of human engraftment and lineage composition were determined in the BM and PB. Briefly, BM cells were harvested by flushing tibiae and femurs with 1× PBS and passing through a 40-μm strainer. Mononuclear cells were blocked with Fc blocking solution (BioLegend) and stained with the following antibody panels; CD45 BV421, CD19 PerCp Cy5.5 (clone HIB19, BioLegend), CD33 FITC (clone P67.6, BD Bioscience), and CD3 PE (clone OKT3, BioLegend) for flow cytometry analysis. To determine human stem cell composition within BM, cells were stained with the following antibody panels: CD45 BV421, CD34 FITC, CD38 APC-Cy7, CD90 PE-Cy7, and CD45RA PerCP Cy5.5.