Cd45ra pb

1 ml viably frozen bone marrow MNCs were thawed in the presence of 100 μl DNAse I (2 mg μl⁻¹) and incubated for 10 min in a solution of 1.6 ml fetal calf serum, 10 μl heparin (5,000 U ml⁻¹) and 100 μl MgSO₄ (0.22 μM). Subsequently, the myeloid progenitor cells were sorted according to a protocol adapted from Pang et al.35 (link) The cells were washed and stained with CD34-APC (Beckman Coulter, Brea, CA, USA), CD38-PE-Cy7 (BioLegend, San Diego, CA, USA), CD123-PE (BioLegend) and CD45RA-PB (BioLegend) monoclonal antibodies. Cells were analysed and sorted using a FACS Aria SORP flow cytometer and DIVA software (Becton Dickinson, Franklin Lakes, NJ, USA). Viable cells were selected based on forward scatter and side scatter profiles, and doublets were discriminated using forward scatter area versus width and side scatter area versus width. The HSC population was defined as CD34⁺CD38⁻. Within the CD34⁺CD38⁺ fraction, the common myeloid progenitor cells (CD123⁺CD45RA⁻), the granulocyte-macrophage progenitor cells (CD123⁺CD45RA⁺) and the megakaryocyte-erythroid progenitor cells (CD123⁻CD45RA⁻) were selected. DNA isolation from these cell fractions, followed by DNA amplification, was carried out using the Qiagen REPLI-g single cell kit (Qiagen) according to the manufacturer’s protocol.