N418, eBioscience), TER-119 (clone Ter119, BD Pharmingen) and CD41 (FITC, clone MWReg30, BD Pharmigen); c-Kit (PerCP-Cy5.5, clone 2B8, eBioscience), Sca-1 (PE-Cy7, clone D7, eBioscience), CD150 (PE, clone TC15-12F12.2, BioLegend) and CD48 (biotin, clone HM48-1, BioLegend). The samples were incubated for 15 min at 4 °C and washed with 2 ml buffer. The cell pellets were resuspended in 200 μl staining buffer containing streptavidin-BV421 and incubated for another 15 min at 4 °C. Finally, cells were washed, resuspended in 500 µl staining buffer, data were acquired on a Fortessa FACS analyser (Becton Dickinson) and analysed with FlowJo v10.0.7. LKS cells were defined as lineage− CD41−Sca-1+ Kit+ and HSCs were defined as LKS CD48− CD150+.
