IEC and IEL Isolation

Intestinal IEL isolation was performed according to the protocol as described previously by our laboratory (13, 32). Mesenteric fat and Peyer's patches were carefully removed. The small intestine or colon was opened longitudinally, washed in RPMI 1640 Medium, and cut into 5 mm pieces. Then, the pieces were incubated in extraction buffer (5 mM EDTA, 2 mM DTT, 10% fetal bovine serum in PBS) with continuous brisk stirring at 37°C for 30 min. The tissue suspension was filtered rapidly through a 40-μm strainer to remove debris and centrifuged at 2,000 rpm at 4°C for 5 min. Pelleted cells were suspended in 40%/70% isotonic Percoll (Sigma, USA) and centrifuged at 2,000 RPM at room temperature for 20 min. The supernatants contained an enriched IEC population. The IECs layer was carefully sucked off and captured for RNA extraction. IELs are at the 40–70% interface. The cells were washed twice and resuspended in RPMI 1640. The viability of the IELs exceeded 95%, according to trypan blue exclusion staining.