Briefly, 1 ml effluents obtained during the last 3 days of each fermentation period from proximal (R1), transverse (R2) and distal (R3) colon reactors were applied directly in duplicate on cell layers of three consecutive passages and incubated at 37°C for 90 min. To kill non-invading bacteria, cell layers were washed twice with 250 μl PBS before adding 250 μl DMEM supplemented with 150 μg/ml gentamicin (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) per well followed by an additional incubation period for 60 min at 37°C. After a further washing step with PBS, 250 μl Trypsin-EDTA (1X, Invitrogen) were added followed by another incubation for 10 min. Finally, cells were disrupted by adding 250 μl 0.1% (V/V) Triton X-100
(Sigma) per well and incubating for 10 min before samples were collected GSK690693 mouse for enumeration of buy PF-6463922 invaded Salmonella. The same protocol but without gentamicin treatments was used for the determination selleckchem of cell-associated Salmonella (accounting for both invasive and adherent bacteria). The number of adhered Salmonella was then calculated from the difference of cell-associated to invaded bacteria. Adhesion and invasion ratios were expressed
as the percentage of adhered and invaded bacteria, respectively, related to the total number of Salmonella present in effluents. Invasion efficiency measured during different probiotic and prebiotic treatments was expressed as the percentage of invaded bacteria related to the number of cell-associated Salmonella. The same protocol was used to measure the invasion efficiency of S. Typhimurium N-15 in pure culture when applied in artificial DMEM medium. Therefore, the pellet of an overnight culture of Salmonella obtained by centrifugation (8000 g, 5
min) was diluted in DMEM to reach a concentration Nintedanib (BIBF 1120) of 1.0 × 107 cfu/ml. 125 μl of this bacterial suspension was added in duplicate to cell monolayers that corresponded to a Salmonella concentration (1.3 × 106 cfu/ml) measured in effluents from the two models during Sal periods. Transepithelial electrical resistance (TER) measurements TER measurements were performed to estimate the degree of cell monolayer’s integrity loss that occurs during Salmonella infection due to disruption of tight junctions . To measure the epithelial integrity of HT29-MTX cells, 400 μl of effluent was applied directly to the apical compartment of PBS-washed HT29-MTX cell culture inserts that were prepared as previously described. TER measurements were performed before effluent application and after 1, 2, 3 and 24 h of incubation at 37°C. The resistance of cell layers was calculated by subtracting the intrinsic resistance of the filter insert alone from the total measured resistance (filter insert plus cell layer and effluents) and expressed as Ω per cm2 surface area. The same protocol was used to measure the influence of S. Typhimurium N-15 on TER of HT29-MTX cells in artificial DMEM medium as presented before.