GG, heat-killed L.GG or its conditioned medium preserve the intestinal epithelial barrier, after disruption with gliadin? c) what are their effects on the TJ protein expression? The role of cellular polyamines as a requisite for L.GG action on the expression of TJ proteins was also investigated. As in vitro model of CD the Caco-2 cell line was used. This line is formed by intestinal epithelial cells obtained from human colon adenocarcinoma, that, before confluence, mimics the physiological enterocytes, and provides an important and widely used tool for studying and obtaining greater insight into the molecular and cellular
mechanisms of CD alterations in epithelial cells . Methods Cell culture conditions Human colon adenocarcinoma-derived Caco-2 cells were obtained from the Interlab Cell Line Collection (IST, Genoa, Italy). Cells were routinely cultured
in RPMI-1640 medium supplemented with AZD8186 order 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, MLN8237 100 μg/ml streptomycin, in a monolayer culture, and incubated at 37°C in a humidified atmosphere containing 5% CO2 in air. At confluence, the grown cells were harvested by means of trypsinization and serially subcultured with a 1:4 split ratio. All cell culture components were purchased from Sigma-Aldrich (Milan, Italy). OICR-9429 supplier bacterial strain As probiotic, the Lactobacillus rhamnosus ATCC 53103 (commercially named Lactobacillus GG, L.GG, obtained from the American Type Culture Collection ATCC, Manassas, VA USA) was tested in our set of experiments. L.GG was cultured at 37°C for 24 h under anaerobic conditions in Man-Rogosa-Sharpe (MRS) broth; the incubate was centrifuged (300 × g for 10 min) at room temperature and the Urease precipitate was collected and washed twice with phosphate buffered saline (PBS) at pH 7.4. The bacteria were then re-suspended in RPMI-1640 medium in order to give a bacterial concentration of 108 CFU/ml (as determined by
colony counts). Heat-treatment of L.GG was performed by heating at 95°C for 1 h. Bacterial conditioned medium (CM) was collected by centrifugating the incubate at 300 × g for 10 min. The supernatant (conditioned medium) was filtered through a 0.22 μm low-protein-binding filter (Millex; Millipore, Bedford, MA) to sterilize and remove all bacterial cells. Aliquots of L.GG-CM were stored in sterile microcentrifuge tubes at −20°C until use. Caco-2 cells were treated with LGG-CM as a 10% volume of the total incubation cell medium. Gliadin and L.GG treatments Caco-2 cells (25th-30th passage) were seeded at a density of 2 × 105 cells/5 ml of supplemented RPMI-1640 in 60 mm tissue culture dishes (Corning Costar Co., Milan, Italy). After 24 h, to allow for attachment, the medium was removed and RPMI-1640 supplemented with 10% FBS and 2 mM glutamine, containing viable L.GG (108 CFU/ml), L.GG-heat killed (L.GG-HK), L.GG-CM were added to cells for 6 h.