Intestinal epithelial cell isolation

Intestinal epithelial cells (IECs) were isolated using previously published protocol (Zeineldin and Neufeld, 2012). Briefly, the intestine was dissected, and contents were cleaned by flushing with cold PBS. Tissues were then incubated in 0.04% sodium hypochlorite to remove bacterial contaminants, then rinsed with PBS before incubation in buffer containing DTT and EDTA. After 15 min incubation on ice, intestinal pieces were transferred to PBS and vortexed to dissociate epithelial layer cells. Incubation and mechanical dissociation were repeated twice more. Isolated cells were centrifuged onto a microscope slide using a cytocentrifuge, and IF staining with EpCAM Ab was performed to confirm the isolation of epithelial cells (data not shown). Trizol was used to prepare RNA from obtained cells.