Isolation of Intestinal Immune Cells

Cells from intestinal lamina propria, Peyer’s patches, and mesenteric lymph nodes were isolated as described previously (Rios et al., 2016 (link)). Briefly, Peyer’s patches and mesenteric lymph nodes were mechanically dissociated in ice-cold PBS. The resulting cell suspensions were passed through a 70-um mesh cell strainer. To prepare the intestinal lamina propria cells, associated fat and Peyer’s patches were removed, the intestinal tissue was washed in ice-cold PBS to remove the luminal contents and cut open longitudinally, and the tissue was cut into four equal-sized pieces. Epithelial cells were removed by shaking the tissues in PBS with 1 mM EDTA, 1 mM dithiothreitol, and 10% fetal calf serum for two rounds of 20 min at 37°C. Then, the pieces were washed three times with PBS to remove the EDTA, minced exactly 40 times in a microfuge tube, and incubated in 20 ml of RPMI-1640 supplemented with 1.5 mg ml^-1 Collagenase II (Biosharp), 2.5 mg ml^-1 hyaluronidase (Biosharp), and 0.25 mg ml^-1 DNase I (Solarbio) for 45 min at 37°C with constant shaking. Cell suspension was then extracted by passing the tissue and supernatant over a 70-µm mesh cell strainer. The cell suspension was then centrifuged, and the resuspended pellet was further purified from the interface of a 45/72% Percoll density gradient.