Cd45.1 percp cy5 5 a20

For WEHI-231 apoptosis analysis, the active Caspase 3 kit (C92-605, BD Pharmingen) was used following the manufacturer’s instructions. Briefly, cells were fixed and permeabilized with Fix/Perm Buffer for 30 min at 4°C, stained with an antibody for active Caspase 3 labelled with APC for 30 min at 4°C, and analyzed by flow cytometry. For splenic B cell analysis of IgM^b-macroself mice reconstituted with retrovirus-transduced HPSCs or bone marrow cells from donor mice, single-cell suspensions were obtained by disaggregating spleens through a 0.70μm cell strainer, depleted of red blood cells by incubation for 5 min RT with ACK lysis buffer, stained with CD45.1-PerCP/Cy5.5 (A20; Biolegend), CD45.2-PE (104; Biolegend), IgM-APC (RMM-1; Biolegend) and CD19 PE/Cy7 (1D3, eBioscience) for 20 min at 4°C, and analyzed by flow cytometry. Data were acquired on FACS Calibur, Novocyte or Fortessa X-20 flow cytometers, and analyzed with Flowjo Version10.7.1.

Recipient mice were irradiated at 800rad in an X-ray irradiator and placed on antibiotics by diluting 6mL of Pediatric Suspension Cherry Flavor (NDC 65862-496-47) into 250mL water bottles. Approximately 16 hours later, bone marrow was harvested from donor mice by flushing the marrow from femurs, tibias, and humeruses with 2.5% FBS in PBS. The cell suspension was run through 70μm cell strainers and red blood cells lysed by treating with ACK lysis buffer for 2min at room temperature before lysis was stopped upon addition of further 2.5% FBS in PBS. Cell suspensions were washed twice with PBS before being suspended in PBS for injection via tail vein. Recipient mice received 6x10⁶ donor bone marrow cells in 0.2mL total volume. Mice were kept on antibiotics for 6 weeks. To assess chimerism via congenic markers, blood was collected via retro-orbital bleeds from recipient mice at week 8, RBCs lysed with ACK lysis buffer as described above, and samples stained for flow cytometry. The markers used were CD8-BV785 (53–6.7, BioLegend), CD4-BV786 (RM4-5, BD Biosciences), CD11b-FITC (M1/70, Tonbo), CD11c-PECy7 (N418, Biolegend), B220-FITC (RA3-6B2, eBioscience), CD45.1-PerCPCy5.5 (A20, BioLegend), and CD45.2-PE (104, eBioscience).