Isolation and Sorting of Intestinal Epithelial Cells

The epithelial fraction of the small intestine was isolated as described previously^{9 (link)}. Briefly, mice were euthanized, and small intestine was collected in cold PBS. Tissues were washed with cold PBS twice, chopped and transferred to new tubes followed by incubation in stripping buffer (10% FBS, 15 mM HEPES, 5 mM EDTA, in 1X HBSS) for 20 min at 37°C. The tissues were vortexed for 15 sec and passed sequentially through 100 microns and 40 microns filters. The collected cells were washed with cold PBS and stained with live/dead staining kit (live/dead fixable blue dead cell stain kit, Life Technologies) according to manufacturer’s protocol. After live/dead staining, cells were stained with anti-CD24 (eBioscience; clone 30-F1), anti-CD45 (Biolegend, clone ), anti-UEA-1 (Vector labs), anti-cytokeratin 18 (Abcam; clone C-04). Goblet cells were sorted using BD ARIA II cell sorter (BD Biosciences) by gating on CD45⁻CD24⁻CK-18⁺UEA-1⁺, while the remaining IECs were collected by gating on CD45⁻CD24⁻CK-18⁻UEA-1⁻ and collected in RPMI containing 10% FBS^{21 (link)}. The collected cells were centrifuged and resuspended in Tri Reagent (Invitrogen) for RNA extraction. Data were processed using FACSDiva software (BD) and FlowJo (FlowJo).