M1 69

Mature CD24⁻ thymocytes were isolated by Dynal negative selection using biotinylated anti-CD24 mAb M1/69 (Ebioscience). Cells were labeled with 10 μM CFSE and injected intravenously to CD45.1 recipients (1×10⁶/mouse).

SI and colons were harvested from mice, flushed with PBS, cut open longitudinally, and incubated with EDTA and 1 mM DTT for 30 minutes at 4C on a rocker. For RNA experiments 3 mM EDTA was used and for flow cytometry 30 mM EDTA was used. Supernatants were then passed through a 100 micron filter and centrifuged at 650×g for 5 minutes. For RNA, IECs were then resuspended in RNAzol, homogenized by passing repeatedly through a 25-gauge needle, and stored at 80C until RNA was isolated. For flow cytometry, IECs were further digested in 0.3 U/mL Dispase I (Roche) and 1 mg/mL DNAase I (Sigma) for 10 minutes at 37C. IECs were then centrifuged at 650×g for 5 minutes and resuspended in flow cytometry buffer (PBS plus 3% fetal calf serum, 1mM EDTA, and 0.05% azide) before treating with antiCD16/32 antibodies (UCSF Antibody core) and staining with antibodies to Epcam (ebioscience clone G8.8), CD45 (ebioscience clone 30-F11), and CD24 (ebioscience clone M1/69). 4’,6-diamidino-2-phenylindole (DAPI, Thermo-Fisher) was used to exclude dead cells. Before running, IECs were passed through a tube-top 35 micron filter. Data were collected on a LSR Fortessa (BD Biosciences) and analyzed using FlowJo software. Purity of IEC isolations was repeatedly assessed for RNA experiments and was consistently 95–99% CD45^-IECs.