After this time, E. coli strains (150 μL of an overnight culture) were added to the well to begin the coinfection period that lasted 2 h. The coverslips were washed, fixed and stained as described above. Quantitative mixed infection assays were carried out counting the colony-forming units (CFU). The infection steps were performed in the same way as described for the qualitative assays. After the coinfection step, wells were washed five times with D-PBS and HeLa cells and bacteria were suspended in 0.5% Triton X100 in PBS (2 mL/well). The suspension was then diluted 1:1000 in PBS and 50

μL of the suspension was plated onto MacConkey agar plates. CFU counting was carried out determining the number of lactose-fermenting colonies (E. coli) and non-fermenting colonies (C. freundii). At least three independent assays were performed with each bacterial Salubrinal in vitro suspension plated in triplicate. Adhesion assays using pre-conditioned Selleck Veliparib medium Pre-conditioned DMEM-mannose media were used to verify the role of chemical

signals in the studied events. Employing polycarbonate permeable inserts (Transwell®-Corning) with high pore density (108 per cm2) the plate wells were separated into two compartments; the upper compartment was loaded with 100 μL of DMEM-mannose, and the lower compartment with 400 μL. The medium was pre-conditioned inoculating the upper compartment with 100 μL of overnight bacterial www.selleckchem.com/products/ro-61-8048.html culture for two hours at 37°C. Thereafter, HeLa cells, in the lower compartment, were infected with 150 μL of 18-h culture of the tested bacteria for two hours at 37°C. Finally, the cells were washed, fixed and stained as described above. Pre-conditioned HeLa cells Adherence assays were also Bay 11-7085 performed employing HeLa cells pre-conditioned

by the initial adhesion of C. freundii strains. Briefly, host cells were pre-infected with an overnight culture (50 μL) for two hours, treated with gentamicin [200 μg/mL] for one hour, and then washed several times with D-PBS in order to remove the adherent bacteria. Afterwards, pre-conditioned HeLa cells were employed to test the adhesion of EAEC strains using 150 μL of an overnight culture for two hours at 37°C. Bacterial aggregation assay Five milliliters of DMEM-mannose in 10 mL test tubes were inoculated with 10 μL of overnight bacterial cultures (or 5 μL of each bacterial culture when the bacterial aggregation of cocultures were tested) and incubated at 37°C for 18 h without shaking. Afterwards, the optical density at 600 nm (OD600 nm) of the culture upper layer (700 μL) was determined for the standing and homogenized culture. Bacterial aggregation was evaluated using the following rate: 1- (standing culture OD/homogenized culture OD). Bacterial settling profile In order to verify the differences in bacterial aggregation, settling assays were carried out to follow bacterial settling kinetics. Settling assays were conducted with DMEM-mannose (1.