Matrigel coated

To study the translocation of β-glucan through human FAE, an in vitro coculture model was used. Coculture of Peyer’s patch lymphocytes and intestinal epithelial cells can trigger epithelial cell conversion to an M cell-like phenotype,^{37 (link)} and we previously established a modified version of this coculture.^{14 (link)} Briefly, intestinal epithelial Caco-2-cl1 cells (originally obtained from Dr. Maria Rescigno, Italy) were grown for 14–17 days on Matrigel-coated (Becton Dickinson, USA) 3.0 μm polycarbonate filters (Costar, Baedvenhorp, NL) until reaching confluence. The model FAE was obtained by adding 5x10⁵ Raji B cells (ATCC, ML, USA) suspended in DMEM-supplemented media to the basolateral chamber of confluent Caco-2-cl1 monolayers. Corresponding monocultures of Caco-2-cl1 cells on matched filter supports served as controls. The coculture was maintained for 4–6 days until M cells were generated. To confirm transformation to M cells, TER was measured daily, and to be considered transformed, TER should be at least 10% lower in the coculture model compared to the monoculture.^{14 (link)}

The Matrigel-coated (Becton Dickinson, Franklin Lakes, NJ, USA) (or not) Transwell chambers (Corning Life Sciences, Corning, NY, USA) were employed for invasion (or migration) assays. The upper chamber was set with cells (1 × 10⁴) and RPMI-1640 medium without FBS, and the lower chamber was set with RPMI-1640 medium with 10% FBS. Next, the invaded or migrated cells were fixed through 90% ethanol and dyed with 0.1% crystal violet. The invaded or migrated cells were examined through the inverted microscope (IX71, magnification ×200, Olympus, Tokyo, Japan). Images were analyzed using ImageJ (National Institutes of Health, NIH, Bethesda, Maryland, USA).

In vitro invasion of HTR8/SVneo cells was measured in Matrigel-coated (Becton Dickinson; Franklin Lakes, NJ) transwell inserts (Costar, Cambridge, MA) containing polycarbonate filters with 8-µm pores as previously described [37] (link). The inserts were precoated with 50 µg Matrigel matrix in accordance with the manufacturer's recommendations. Briefly, The cells were pretreated with a proliferation inhibitor, mitomycin C (Sigma-Aldrich Corp.), 10 µg/ml, for 2 hours. 1×10⁵ cells per well were plated into the upper chamber in 200 µl RPMI 1640 medium without FBS. 800 µl of medium with 10% FBS was placed into the lower well of the chamber. After 24 hr, the remaining cells on top of the transwell were removed with a cotton swab. The filters with invaded cells attached were washed with PBS, fixed in methanol for 10 min, and stained with hematoxylin. Finally, the number of invaded cells was counted under a light microscope in 15 random-selected non-overlapping fields from each chamber at a magnification of 200×. Average cell numbers in each field were used for statistical analyses. Each experiment was performed in triplicate. All experiments were conducted in triplicate and the invasion index was expressed as the percentage of invaded cell number compared with the corresponding control.