Single HSCs, defined as lineage−c-Kit+Sca-1+CD41−CD48−CD150+, were sorted into 100 μl StemSpan SFEM medium (StemCell Technologies) in each well of a round bottom, 96-well plate (Costar) using a Synergy cell sorter (Sony Biotechnology). The plates were spun down for 5 min at 180g and the presence of a single cell per well was confirmed visually. The full content of selected wells was loaded into insulin syringes (29G, 0.5-inch needle) containing 2 × 105 carrier cells in 300 μl Hank’s balanced salt solution (StemCell technlogies). The contents of the syringe were used to dislodge the single HSC from the bottom of the well, avoiding the creation of bubbles. The entire volume (400 μl) was injected into the tail veins of irradiated recipients and chimaerism was measured every 2 weeks using flow cytometry. Recipients were considered reconstituted by the single stem cell if chimaerism for CD45.2+ WBCs was ≥0.1%.
