Mammalian culture conditions

HT29-MTX cells were obtained from Millipore-Sigma (#12040401). Cells were maintained in Gibco Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) in a humidified atmosphere at 37 °C, 5% CO_2. Cells were tested for Mycoplasma using the Mycoplasma Detection Kit (Lonza, cat# LT07–518). For adhesion assays, HT29-MTX cells were seeded at 2 × 10⁵ cells on poly-L-lysine coated round coverslips and incubated for 3–5 days until confluent. When monolayers were confluent, HT29-MTX cells were incubated with Hoechst 33342 staining dye solution (Invitrogen) in PBS for 10 min at 37 °C, washed, and treated with 1 × 10⁷ cells of CFDA-tagged B. dentium for 1 h aerobically at 37 °C. After the incubation, non-adhered cells were removed with 3x washes of PBS and cells were fixed with Clarke’s Fixative to maintain the mucus architecture. A subset of cells were used for Scanning Electron Microscopy (SEM) imaging using a FEI XL-30FEG microscope. Cells that were reserved for immunostaining were permeabilized with 0.1% Triton-X for 30 min at room temperature, blocked with PBS containing 10% donkey serum, and incubated with an anti-human MUC2 antibody (Santa Cruz, cat # sc-515,032; 1:200 dilution) overnight at 4 °C. Following PBS washes, cells were incubated with donkey-anti-mouse AlexaFluor 555 (Life Technologies, cat # A11004; 1:1000 dilution) for 1 h at room temperature. Coverslips were mounted to slides using FluoroMount (Thermo Fisher Scientific) and slides were imaged on the Nikon Eclipse TiE inverted microscope.