Isolation of mouse lymphocytes

Single-cell suspensions were prepared from lung, liver, spleen, intestinal epithelium (including small intestine and colon), blood, and bone marrow.

For lung and liver, mice were euthanized and lung and liver were perfused with cold PBS. Afterward, tissues were dissected into fragments of 1 mm diameter, digested in RPMI-1640 medium containing 1 mg/ml of collagenase type IV enzymatically digested at 37 °C for 30 min in 5 ml of digestion buffer. The digested tissues were then successively passed through 70 µm sterile nylon filter to enrich for leukocytes and washed with 10 ml of serum-free RPMI-1640 medium. Single-cell suspension was transferred to a 15-ml conical tube and centrifuged for 5 min at 1200 rpm, 4 °C, and supernatant was discarded. The cell pellet was resuspended in 5 ml of 40% Percoll, and centrifuged at 2000 rpm for 15 min at 4 °C with free-brake and low-accelerator conditions. Red blood cells were removed by RBC lysis solution. At last, the cell pellet was resuspended in 1 ml PBS containing 2% FBS and count viable nucleated cells in a hemacytometer using trypan blue dye exclusion.

For spleen, peripheral blood and bone marrow, spleens were isolated from mice and mechanically disrupted in RPMI-1640 medium containing 2% FBS using frosted glass slides to generate a single-cell suspension. Then the cells liquid was passed through 70 µm sterile nylon filter to enrich for leukocytes and washed with 10 ml of serum-free RPMI-1640 medium. Single-cell suspension was transferred to a 15-ml conical tube and centrifuged for 5 min at 1200 rpm, 4 °C, and supernatant was discarded. Red blood cells were removed by RBC lysis solution. At last, the cell pellet was resuspended in 1 ml PBS containing 2% FBS and count viable nucleated cells in a hemacytometer using trypan blue dye exclusion.

Peripheral blood was collected by fundus venous plexus with EDTA as anticoagulant, and washed by PBS containing 2% FBS. Then, the steps after transferring from the cell suspension to the 50-ml tube were the same as the extraction of splenic lymphocytes.

Bone marrow was removed from femurs, tibiae, and hips by flushing with RPMI-1640 medium containing 2% FBS. Then, the steps after transferring from the cell suspension to the 50-ml tube were the same as the extraction of splenic lymphocytes.

For small intestine and colon, in brief, Peyer’s patches and fat tissue were carefully removed. The small intestine and colon were opened longitudinally, washed in RPMI-1640 Medium, and cut into 5 mm pieces, which were transferred to a 50 ml conical tube (Falcon 2070) containing 25 ml EDTA-RPMI medium including 10 mM/L HEPES, 25 mM/L NaHCO₃, 1 mM/L EDTA, 1 mM/L DTT and 2% FBS, and 40 μg/ml penicillin and streptomycin. The tube was shaken at 37 °C for 30 min (horizontal position; orbital shaker at 280 rpm). Cell suspensions were collected in a new 50 ml conical tube and passed through a 70 μm strainer to deplete cell debris and sticky cells (crude cell preparation) and centrifuged at 1200 rpm at 4 °C for 6 min. Cell pellets were resuspended with EDTA-RMPI medium, filtered through 70 μm nylon mesh, and then centrifuged at 1200 rpm at 4 °C for 6 min. Subsequently, the pelleted cells were suspended in 40% Percoll. Percoll was diluted with PBS to make 1x Percoll. Cells were suspended with 16 ml of 40% Percoll in 15-ml tubes. Two milliliters of 75% Percoll was added into the lower layer of 40% Percoll in each 15mltube. IELs were collected by harvesting the center layer between 40 and 70% Percoll after being centrifuged at 2000 rpm for 15 min at 20 °C with free-brake and low-accelerator conditions.