Im modular applications system

The lactobacilli adhesion to immature Caco-2 cells (in vitro biomodelling of small intestinal enterocyte immaturity in preterm infants) and adhesion to immature HT-29 cells (in vitro biomodelling of large intestinal colonocytes immaturity in preterm infants) were performed as described in [18] (link) in modification. Briefly, suspensions of lactobacilli in antibiotic-free medium at 2x10 8 CFU/ml were added to monolayers of immature Caco-2 cells with a layer of 80-100% at MOI 200:1 bacterial cell/epithelial cell ratio. Suspensions of lactobacilli were added to monolayers of immature HT-29 cells at the same conditions. Co-cultures were incubated for 2 h at 37°C in 5% CO 2 atmosphere. After 2 h, co-cultures were washed three times with sterile PBS to remove unbound bacteria, fixed with methanol, stained with azure-Journal Pre-proof J o u r n a l P r e -p r o o f eosin (Pan Eco, Russia) and examined under the Leica DM 4500B microscope (Leica, Canada). Adherent bacteria quantified by using the Leica IM modular applications system (Leica, Canada). Adhesion of lactobacilli cells to epithelial cells was expressed as a percentage of 100 randomly selected epithelial cells with adhering bacteria and as average number of adhering bacteria per epithelial cell.

To assess the effect of Slp2 from LC2029 on adhesion of proteobacteria to monolayers of Caco-2 cells, 5×10 7 cells/mL suspensions of strains Escherichia coli ATCC E 2348/69, E. coli ATCC 31705, Salmonella Enteritidis ATCC 13076, Campylobacter jejuni ATCC 29428, Pseudomonas aeruginosa ATCC 27853 were incubated in the presence of Slp2 at concentrations of 0, 10, 50, 100 µg/mL in phosphate buffered saline (PBS) for 30 min. The strains treated with Slp2 were then applied to the monolayers of Caco-2 cells. The plates were incubated for 1 h at 37°C under 5% CO2. Caco-2 cell monolayers were washed three times with sterile PBS to remove unbound bacteria and Slp2, fixed with methanol, stained with azure-eosin (Pan Eco, Russia) and examined under Leica DM 4500B microscope (Leica, Canada). Adherent bacteria were quantified using Leica IM modular applications system (Leica, Canada). Adhesion of bacterial cells to epithelial cells was expressed as average number of adhered bacteria per epithelial Caco-2 cell.