The ILC2 differentiation potential of hematopoietic progenitors was performed as previously reported (Wong et al., 2012 (link)). In brief, freshly sorted CLPs from the Bcl11bflox/floxRosa26CreERT2/CreERT2 and control Bcl11b+/+ Rosa26CreERT2/CreERT2 mice that were treated with Tam 4 d earlier (4.0 mg Tam by intraperitoneal injection over three consecutive days) were cultured on OP9-DL1 monolayers in the presence of 10 ng/ml IL-7 (PeproTech) and 10 ng/ml IL-33 (PeproTech) for 22 d.
For the short-term fate assay, purified Bcl11b−ChILPs and Bcl11b+ChILPs were cultured on OP9 monolayers in the presence of 25 ng/ml IL-7 (PeproTech) and 25 ng/ml Stem Cell Factor (SCF; PeproTech) for 6 d as previously described (Constantinides et al., 2014 (link)).
For deleting Bcl11b in ILC2s, sorted ILC2Ps from the BM of Bcl11bfloxfloxRosa26CreERT2/CreERT2 and control mice were cultured on OP9-DL1 monolayers in the presence of 20 ng/ml IL-7, 20 ng/ml SCF, and 10 ng/ml IL-2 or 20 ng/ml IL-7 plus 20 ng/ml IL-25 or plus 20 ng/ml IL-33. After 3–5 d, Tam was added in the medium to induce Bcl11b deletion in vitro. Cells were collected and analyzed 10–14 d after Tam treatment.
For the short-term fate assay, purified Bcl11b−ChILPs and Bcl11b+ChILPs were cultured on OP9 monolayers in the presence of 25 ng/ml IL-7 (PeproTech) and 25 ng/ml Stem Cell Factor (SCF; PeproTech) for 6 d as previously described (Constantinides et al., 2014 (link)).
For deleting Bcl11b in ILC2s, sorted ILC2Ps from the BM of Bcl11bfloxfloxRosa26CreERT2/CreERT2 and control mice were cultured on OP9-DL1 monolayers in the presence of 20 ng/ml IL-7, 20 ng/ml SCF, and 10 ng/ml IL-2 or 20 ng/ml IL-7 plus 20 ng/ml IL-25 or plus 20 ng/ml IL-33. After 3–5 d, Tam was added in the medium to induce Bcl11b deletion in vitro. Cells were collected and analyzed 10–14 d after Tam treatment.
