Szx12 dissecting scope

For intestinal lamina propria lymphocyte preparations, intestines were isolated, attached fat removed and tissues cut open longitudinally. Luminal contents were removed by shaking in ice-cold PBS (Corning). For AOM/DSS protocol, distal luminal polyps were excised from the colon with an Olympus SZX12 dissecting scope. Afterwards, mucus was gently removed by forceps. Epithelial cells and intraepithelial lymphocytes were then removed by shaking tissue in HANKS free media (Sigma-Aldrich) containing 2% FBS (Omega Scientific) and 5 mM EDTA (Thermo Fisher Scientific) stripping buffer for 30 min at 37 °C. Samples were then vortexed and the epithelial fraction was discarded. Afterwards, samples were washed by PBS and enzymatic digestion was performed in RPMI containing 10% FBS and 0.4 U/ml dispase (Thermo Fisher Scientific), 1 mg/ml collagenase III (Worthington) and 20 μg/ml DNase I (Sigma Aldrich) on a shaker for 45 min at 37 °C. Leukocytes were filtered through a 70 μm cell strainer and further enriched by a 40% Percoll gradient centrifugation (GE Healthcare).

The DSS and AOM/DSS mouse models of chronic colitis and CRC, respectively, were established using the protocol previously described (Thaker et al., 2012 ; Wirtz et al., 2017 (link)) with slight modifications. In brief, for the development of chronic colitis, mice were given 3% DSS (MP Biochemicals, colitis grade) in drinking water for 7 days, followed by drinking water for 14 days. This cycle was repeated once. For CRC, mice were given an initial intraperitoneal injection of 12.5 mg/kg AOM and DSS treatment was then started at day 5 after AOM injection. DSS cycle was repeated once for MHCII^∆ILC3 and littermate controls or twice for C57BL/6J mice. Mice were sacrificed, stool and tissue were collected, and colons were examined macroscopically for tumor analyses, then fixed in formalin for paraffin embedding and histology (Bialkowska et al., 2016 ). Macroscopically visible colon adenomas were quantified with an Olympus SZX12 dissecting scope and sized with a digital caliper.