2.3. Lymphocyte isolation and staining
Mice lymphocytes from intestines were isolated as described previously.17,18 Pieces of ileum, 3 cm in length, were cleaned off feces by flushing with a syringe containing 10 ml PBS. Cleaned pieces were cut with a scalpel imnto 0.5 cm pieces. The ileum pieces were predigested with 10 ml Pre-digestion buffer (Phosphate-buffered saline: PBS + Ethylenediaminetetraacetic acid: EDTA) at 37 degrees for 20-30 minutes (shaker incubator). Supernatant were removed. Ileal pieces were transferred into 10 ml Digestion solution (collagenase, DNase, complete RPMI) and the SI tissues were further cut into 1mm pieces with a scalpel. Then, SI tissues were incubated at 37 degrees for 45-60 minutes via shaker incubator. After 1 min of vortexing, the cells were passed through the 70 µm cell strainer. The cells were centrifuged (400 g) for 5 minutes and the supernatant was discarded. The pellet was resuspended in 5 ml 40% percoll and overlayed on 5 ml 90% percoll.The percoll gradient was centrifuged for 20 minutes at 400 g w/o brakes. The lymphocytes at the interphase was collected, counted by trypan blue.
Staining was performed in round bottom 96-well plates. Fc-block was added and incubated for 5 minutes. Then, ILC staining antibodies were added according to manufacturer’s dilution recommendation. After 30 minutes of incubation in dark and on ice, cells were washed with staining buffer twice, spun at 400 g. Then, cells were resuspended in 200 μl staining buffer and run on FACSAria III.17,18Antibody list: Alexa Fluor 647 Anti-Mouse NK1.1 (clone: PK136), APC Anti-Mouse CD11b (clone: M170), APC Anti-mouse CD3 (clone: 17A2), PE/Cy7 Anti-Mouse CD90 (clone: 30H12), PerCP/Cy5.5 Anti-Mouse CD45 (30F11), FITC Anti-Mouse B220 (RA3-6B2).