Ld depletion column

For in vivo differentiation experiments, CHILPs were highly purified by magnetic beads enrichment followed by FACS sorting. Briefly, single-cell suspension of leukocytes obtained from bones was stained with biotinylated antibodies against CD11b (M1/70, eBioscience), CD19 (MB19-1, Biolegend), TER-119 (TER-119, eBioscience). Stained cells were passed through LD depletion column (Miltenyi Biotec) according to manufacturer’s instructions. The enriched Lineage negative fraction was stained with fluorescently labeled antibodies (as described above) and then sorted using a BD FACSAria I or BD FACSAriaIII cell sorter (BD Biosciences). Highly purified CLP (Lin^neg CD127⁺ Id2⁻ CD135⁺) or CHILPs (Lin^neg CD127⁺ Id2⁺ CD135⁻ α4β7⁺ CD25⁻) from PBS or B16-Flt3L injected Id2^Gfp/+ donors were injected intravenously into Rag2^−/−Il2rg^−/− recipient mice. Each recipient mouse received between 150 to 2000 cells. Five to ten weeks after transfer, recipient mice were sacrificed and donor-derived cells were analyzed in the indicated organs. Engraftment index was calculated by dividing the number of ILCs retrieved in the host by the number of progenitors cells injected in the same mouse.

Cells were dissociated from the monolayer using TrypLE Express (Gibco) and washed 1X in PBS/10% fetal bovine serum (FBS; Wisent Inc.) (FACS buffer) containing 10 μM Y27632 (Tocris). Live cells were incubated for 20 min at room temperature with an anti-GP2 antibody in FACS buffer. After washing once with FACS buffer, the live cells were incubated for an additional 20 min at room temperature with an anti-mouse PE-conjugated secondary antibody in FACS buffer. The cells were washed once again with FACS buffer, and re-suspended in MACS buffer (Miltenyi Biotec) with anti-PE microbeads (Miltenyi Biotec) at a concentration of 75 μl MACS buffer and 20 μl beads/1 × 10⁷ cells. They were incubated at 4 °C for 15 min, followed by one wash in MACS buffer. The cells were then loaded onto a LS positive selection column (Miltenyi Biotec) and the flow through collected. The column was washed thrice and the cells eluted, all in FACS buffer with 10 μM Y27632 (Tocris). The elution from the LS positive selection column formed the GP2⁺ fraction. The flow through from the LS positive selection column was then loaded onto a LD depletion column (Miltenyi Biotec). The flow through from the LD depletion column was used as the flow through fraction. Following MACS, the cells were cultured as aggregates. Supplementary Table 3 for list of antibodies used.