Anti mouse cd45.1 pe cy7

Eight- to ten-week-old NOD/SCID IL2Rγ^−/− mice were intravenously injected with 1*10⁵ MOLM-13 cells. Beforehand, AML cells were cultured alone or in the presence of either purified ECAR or control vector modified T cells at an E:T ratio of 5∶1 for 4 h. Prior to the experiment, T cells were irradiated at 5 Gy to prevent xenograft reaction in recipient mice [50] . Mice were sacrificed when visible tumors developed at injection site and single-cell suspensions from bone marrow obtained from femur and tibia of the left hind leg were prepared. Erythrocytes were removed by lysis and nucleated cells were stained with anti-mouse CD45.1/PE-Cy7 (eBioscience, clone A20), anti-human CD3/APC-eFluor780 (eBioscience, clone SK7), CD19/APC (BD Bioscience, clone HIB19), CD33/PE (eBiosience, clone HIM3-4), and CD45/AlexaFluor700 (Biolegend, clone HI30) mabs. Doublet discrimination was routinely carried out and dead cells were excluded by 4, 6 diamidino-2-phenylindole (DAPI)-staining (Sigma-Aldrich, Taufkirchen, Germany). All measurements were performed on a BD LSRII FACS machine (BD Biosciences). Data analysis was realized using FlowJo-software (Tree Star Inc., Ashland, USA).

Between 14 and 16 weeks post transplantation of edited CD34⁺ HSPCs, mice were euthanized and bone marrow was harvested from tibia, femur, pelvis, sternum and spine using a pestle and mortar. Mononuclear cells (MNCs) were enriched using a Ficoll gradient centrifugation (Ficoll-Paque Plus, GE Healthcare) for 25 min at 2000 g at room temperature. The samples were then stained for 30 min at 4 °C with the following antibodies: monoclonal anti-human CD33 BV 421(1:50 dilution, 6 ul in 300 ul of MNCs pelleted in MACS buffer (1× PBS, 2% fetal bovine serum, 2 mM EDTA); anti-human HLA-ABC FITC (1:100 dilution; W6/32; BioLegend); anti-human CD19 PerCp-Cy5.5 (1:20 dilution; HIB19; BD Biosciences); anti-mouse CD45.1 PE-Cy7 (1:200 dilution; A20; eBiosciences); anti-human CD34 APC (1:50 dilution; 581; BioLegend); Multilineage engraftment was established by the presence of myeloid cells (CD33⁺) and B cells (CD19⁺) in engrafted human cells (mCD45^-, HLA-A/B/C⁺ cells).

Sixteen weeks after transplantation, mice were euthanized and mouse bones were collected and processed as follows. For Figure 1E, 2× femur, 2× tibia, 2× humerus, sternum, 2× pelvis, and spine were collected and crushed using a mortar and pestle. Mononuclear cells were enriched using Ficoll gradient centrifugation (Ficoll-Paque Plus, GE Healthcare) for 25 min at 2,000 × g at room temperature. For samples analyzed at week 8 after transplant, bone marrow aspirates were taken from the right femur for analysis, and for all other studies, femurs were taken from mice and flushed with a 25G needle. Cells were then treated with red blood cell lysis buffer (1× RBC buffer; Invitrogen) for ten minutes. All samples were then blocked (10% v/v; TruStain FcX, BioLegend) and stained for 30 min at 4°C with the following antibodies before analysis: monoclonal anti-human CD45 V450 (HI30; BD Biosciences); HLA-ABC APC-Cy7 (W6/32; BioLegend); CD19 APC (HIB19; BD Biosciences); anti-mouse PE-Cy5 mTer 119 (TER-119; eBioscience); and anti-mouse CD45.1 PE-Cy7 (A20; eBioScience). Multi-lineage engraftment was defined as the presence of myeloid cells (CD33+) and B cells (CD19) within engrafted human cells (CD45+; HLA/ABC+ cells).