Isolation of Immune Cells from Murine Intestine and Lymphoid Organs

Cells from colonic lamina propria (LP), mesenteric lymph nodes (MLN) and spleen were isolated as described previously [22 (link), 27 (link)]. Briefly, MLN and spleen were mechanically dissociated in ice-cold PBS. The resulting cell suspensions were passed through a 70-μm mesh cell strainer, and spleen also needed erythrocyte lysis. To prepare colonic LP cells, associated fat and Peyer’s patches were removed, the colonic tissue was washed in cold PBS to remove fecal contents and cut open longitudinally, and the colon was cut into 2-cm pieces and washed twice in 2 mL PBS containing 1 mM dithiothreitol, 10% FCS and 1 mM EDTA with constant agitation for 40 min at 37 °C. And then the pieces were minced exactly 50 times to obtain 1 mm tissue fragments and incubated in 2 mL of RPMI-1640 supplemented with 2.5 mg/mL Hyaluronidase (Biosharp), 1.5 mg/mL Collagenase II (Biosharp), and 0.25 mg/mL DNase I (Solarbio) with constant agitation for 45 min at 37 °C. The cells suspension was extracted through filtration and centrifugation, and the resuspended pellet was further purified from the interface of a 45%/72% Percoll density gradient.