Epcam bv421

Characterization of adherent cultures required dissociation into single cells by incubation with prewarmed TrypLE-select™ at 37 °C. Incubation time varied with the type of cell culture; 5 minutes for neonatal human thymus-derived TEC cultures and 10-15 minutes for and PSC-derived FOXN1+ cultures. Conjugated monoclonal mouse anti-human antibodies: CD104-APC (1:50, clone 422325, Invitrogen), EPCAM-PeCy7 (1:200, clone 12c2, BioLegend), EPCAM-BV421(1:50, clone 9C4, BioLegend), CD90-PE (1:100, clone 5e10, Biolegend), CD90-BV421 (1:50, clone 5e10, Biolegend) were diluted in FACS wash buffer (PBS supplemented with 5% fetal bovine serum) and incubated with cells for 20 minutes on ice. The cell suspension was washed twice with FACS wash solution to remove unbound antibodies and resuspended in FACS wash solution containing 1 μg/ml propidium iodide. Cell surface staining was examined using a Becton Dickenson (BD) LSRFortessa Cell Analyzer. Flow cytometry data were analyzed using the FlowLogic program (7.2.1, DataNova). Alternatively, cell purification was performed using a BD FACSaria FUSION or Influx cell sorter based on cell surface staining or the expression of a fluorescent reporter. Cells were collected using a 5ml FACS tube containing 0.5ml cold fetal calf serum.

Small intestine segments were washed and incubated in HBSS with 30 mM EDTA for 20 min. Epithelial cells were dissociated mechanically through vigorous shaking followed by incubation in Triple-X (Thermo Fisher Scientific) supplemented with Y-27632 (10 mM, Sigma-Aldrich) and 0.5 mM N-acetyl-L-cysteine at 37°C. Dissociated enterocytes were filtered and pelleted before re-suspended in complete advanced DMEM/F12 media containing 10 mM Y-27632 and 0.5 mM N-acetylcysteine. Cells were stained with fluorochrome-conjugated surface antibodies according to the manufacturer’s instructions. For intracellular staining, cells were fixed and permeabilized for 1 hr at 4°C with the Foxp3/ Transcription Factor Staining Buffer Set according to the manufacturer’s instructions (eBioscience). The following antibodies were used for surface or intracellular staining: CD45 BUV393 (BD Cat#565967), Epcam BV421 (BioLegend, Cat# 324219), p-S6 (S235/236) PerCP e710 (eBioscience, Cat# 46900742), p-mTOR eFluor 660 9S24480 (eBioscience, Cat# 50971842), CD4 BV786 (BD, Cat#563727), IFN-γ BV421 (BD, 563376), Lysozyme 1 (rabbit polyclonal; Dako), and goat anti-rabbit Alexa 488 (Molecular Probes). Cell fluorescence was measured using an LSRII flow cytometer, and data were analyzed using FlowJo software (Tree Star).

Single cells from patient P1 were stained with Sytox Blue viability dye (S34857, Life Technologies) and processed on a FACS Aria I instrument. Cells from P2–P16 were stained with anti-EPCAM-PE (347198, BD Biosciences; 1:50 dilution in ice-cold PBS containing 2% FBS) for 30 min with gentle rotation at 4 °C. Mouse lung single cells were similarly stained but with a cocktail of antibodies (1:250 each) against CD45-PE/Cy7 (103114, BioLegend), ICAM2-A647 (A15452, Life Technologies), EPCAM-BV421 (118225, BioLegend) and ECAD-A488 (53-3249-80, eBioscience). Stained cells were then washed, filtered using 40 μm filters, stained with Sytox Blue (human) or Sytox Green (mouse) and processed on a FACS Aria I instrument (gating strategies for epithelial cell sorting are shown in Supplementary Figs. 1 and 4 for human and mouse cells, respectively). Doublets and dead cells were eliminated, and viable (Sytox-negative) epithelial singlets were collected in PBS containing 2% FBS. Cells were washed again to eliminate ambient RNA, and a sample was taken for counting by Trypan Blue exclusion before loading on 10X Genomics Chromium microfluidic chips.