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).