Flow cytometry and fluorescence-activated cell sorting (FACS)

For flow cytometry analysis, 1 million WBCs or Fetal liver cells were stained for 15 min as indicated in Supplementary Data ⁹. After staining, cells were washed with PBS once and then resuspended in 500 µl PBS for flow cytometry on an LSRII cytometer. Data collection was done with BD FACSDiva software (v8.0.1). The gating strategy for different populations is provided in Supplementary Fig ⁹.

For FACS sorting, mature blood cells were stained with biotin-labeled anti-lineage surface marker antibodies as indicated in Supplementary Data ⁹ for 30 min incubation at 4 °C, followed by lineage depletion on an autoMACS machine under “depletes” mode.

Lineage depleted cells were stained as previously described for 15 min. Then cells were washed once and were resuspended to a final cell concentration of 40 M/ml for FACS on an Arial I/II. Post-sort was performed for double checking of cell populations and for cell number counting. The gating strategy for different populations is provided in Supplementary Fig. ⁹.

FlowJo was used for data plot of flow cytometry and FACS.