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Lamina propria cells were isolated from the first 8 cm of intestine according to isolation protocols described in this manuscript. Optimal digestion was achieved when intestinal segments were excised, cleaned and cut into small pieces. Samples were then washed with 2 mM EDTA/HBSS (Gibco) three times for 10 min at 37° C and 200 rpm in a shaking incubator, followed by three pulse vortexing steps at 2500 rpm (maximum speed) for 3 s after each incubation. After the final EDTA wash step, samples were digested in 10 ml RPMI (Gibco) containing 20% FBS (Gibco), 1 mg/ml Collagenase A (Roche #10103578001, 0.223 U/mg solid) and 0.05 mg/ml DNAse (Roche #10104159001, 2916 Kunitz units/mgL) for 30 min at 37°C and 200 rpm in a shaking incubator, with vigorous manual shaking every 5 min. Digestion was quenched with FACS buffer and samples were passed through a 100 µm and 40 µm cell strainer to obtain a single cell suspension. An illustrated step-by-step protocol describing the procedure can be found in Appendix 1.
Individual duodenum draining mesenteric lymph nodes were identified as the most proximal lymph nodes of the mesenteric lymph node chain (Esterházy et al., 2019 (link); Mayer et al., 2017 (link)), and were digested with 100 µg/mL Liberase TL and 100 µg/mL DNAse I (both from Roche, Germany) for 30 min at 37°C and passed through a 70 µm cell strainer.

Wells (Costar 3422, 24 well, 8um plate) were coated at 4°C overnight with with BME Pathclear Matrigel (Trevigen 3442-005-01; 5 μg/well). Cells were harvested with 5mM EDTA / HBSS (Gibco), and washed 3 x with 10 ml culture medium+ 0.1% BSA. After counting, cells were seeded at 50000 or 25000 cells in 100μl media + pen/strep + 0.1% BSA into the top chamber of Matrigel transwells (triplicate wells for each cell line). For some experiments (Figure 5C), pro-N-cadherin monoclonal antibody [30 (link)] (kindly provided by Dr. James Wahl) or isotype control antibody (mouse IgG1, Sigma) were added with cells to top chambers. 600 μl complete media with serum was added to the bottom chamber of each transwell as a source of chemo-attractant. Plates were incubated at 37°C/ 5% CO₂. After 4 h plates were removed and the tops of the transwell inserts were wiped with a Q-tip to remove cells. The inserts were fixed with cold (-20°C) Methanol for 10 minutes and then stained with 0.2 mg /ml Crystal Violet in 2% Ethanol for 10 min. Inserts were left to air dry overnight and photographed at 100X. The number of invaded cells from 5 fields per insert were counted using cell count in Image J software (NIH). Mean # invasive cells from triplicate wells (+/- SD) was determined for each cell population.