To assess engraftment of single HSCs into irradiated recipients, 50 μl of blood was obtained from the tail vein of recipient mice every two weeks. RBCs were lysed by the addition of 1 ml of ammonium chloride lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2) and incubated for 10 min at room temperature. After centrifugation, the cell pellets were resuspended in 100 µl of staining buffer containing antibodies against: CD4 (FITC, clone H129.19, BD Pharmingen), CD8a (FITC, clone 53-6.7, BD Pharmingen), CD45R/B220 (PerCP-Cy5.5, clone RA3-6B2, BioLegend), CD11b/Mac-1 (PE, clone M1/70, BD Pharmingen), Ly-6G/Gr-1 (PE, clone 1A8, BD Pharmingen), TER-119 (PE-Cy7, clone TER-119, BioLegend), CD45.1 (BV421, clone A20, BioLegend) and CD45.2 (APC, clone 104, BioLegend). After incubation, cells were washed with 3 ml of staining buffer before being resuspended in 250 μl of the same buffer. Samples were run on the Fortessa analyser (BD) with the HTS module and the multilineage chimaerism was calculated using FlowJo v10.0.7. TER-119 was used to exclude RBC debris and chimaerism was calculated for each of the WBC lineages (CD45+ total
