Mouse collagen 4

Caco-2 intestinal epithelial cells (American Type Culture Collection) were seeded for 2 weeks on minimum essential medium (MEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillin/streptomycin (100 U/mL) at 37 °C in 5% CO₂ and moisture to allow cellular differentiation. Caco-2 cells were then seeded onto 20 mm coverslip coated with mouse collagen IV (Trevigen, Gaithersburg, MA, USA) in 12-well plate at 10⁵ cells per well 24 h before being challenged with the bacteria. The adhesion of probiotics strains to Caco-2 cells was evaluated as previously described [21 (link)]. Briefly, the stationary phase lactic acid bacteria (LAB) strains grown in MRS were diluted in MEM to an optical density of 0.9 at 600 nm. An aliquot of this culture was used to challenge a confluent Caco-2 intestinal epithelial cells at a multiplicity of infection of ~100 bacteria to epithelial cell in triplicate. The binding studies were performed at 37 °C, 5% CO₂ and 80% humidity for 1 h. For direct visualization of adherent bacteria, the cells were fixed in 99.8% methanol for 10 min at room temperature then stained with Giemsa stain (Muto Pure Chemicals, Tokyo, Japan) at 1:10 dilution for 20 min. A total of 100 cells were examined under the light microscope and the number of adhered bacteria to each cell was counted in 20 randomly selected fields.