Isolation and FACS-Sorting of Intestinal Epithelial Cells

ECs were isolated as previously described (Haber et al., 2017 (link)), with modifications. Briefly, the ileal, cecal, and colonic tissues of VB12- and PBS-gavaged mice were dissected and washed with cold PBS. The tissues were opened longitudinally and cut into small fragments (2–3 cm in length), followed by incubation with 30 mM EDTA-PBS on ice for 30 min, during which the tissues were shaken vigorously every 8–10 min. Isolated crypts were washed once with cold PBS and dissociated with TrypLE Express (Invitrogen) for 10 min at room temperature. The single-cell suspension was passed through a 70-μm cell strainer and used for FACS. To FACS-sort the cells, cell suspensions were labeled with a cocktail of fluorescent antibodies specific for APC-CD45 (catalog 17-0451-83; Invitrogen), FITC-CD31 (catalog 102406; BioLegend), PE/Cy7-TER-119 (catalog 116222; BioLegend), and PE-EpCAM (catalog 12-5791-83; Invitrogen). Dead cells were excluded using LIVE/DEAD Fixable Violet Dead Cell Stain (Invitrogen). CD45⁻ CD31⁻ TER-119⁻ EpCAM⁺ ECs were sorted using a SONY SH800S Cell Sorter or a CytoFLEX SRT Cell Sorter.