Salmonella association and invasion assays were performed according to Zavala et al. (2016) (link). Caco-2/TC-7 cells that were routinely grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) (GIBCO BRL Life Technologies Rockville, MD. USA), supplemented with 15% heat-inactivated (30 min at 60°C) fetal bovine serum (FBS, PAA, GE Healthcare Bio-Sciences Corp., USA), 1% non-essential amino acids (GIBCO BRL Life Technologies Rockville, MD. USA), and the following antibiotics (Parafarm, Saporiti SACIFIA, Buenos Aires, Argentina): penicillin (12 UI/mL), streptomycin (12 μg/mL), gentamicin (50 μg/mL). Cells were seeded in 24-well culture plates (Corning, NY, USA) at 2.5 × 105cells per well and incubated at 37°C in a 5% CO2 — 95% air atmosphere. Caco-2/TC-7 cells were used at post-confluence after 7 days of culture.
Salmonella enteritidis serovar enteritidis CIDCA 101, provided by Dr. H. Lopardo, was grown in nutritive broth (Biokar Diagnostics, Beauvais, France) for 18 h at 37°C (Golowczyc et al., 2007 (link)). Confluent Caco-2/TC-7 monolayers were washed twice with sterile PBS (pH 7.2). Cells were pre incubated for 1 h at 37°C in a 5% CO2—95% air atmosphere with 250 μL EPS solutions (300, 500 and 800 mg/L in DMEM) or 250 μL DMEM in the case of Salmonella association and invasion controls. Afterwards, 250 μL of Salmonella suspension (1 × 107 CFU/mL) were added to each well and incubated 1 h at 37°C in a 5% CO2—95% air atmosphere. For association assays, cells were washed three times with PBS and lysed with 500 μL/well of bi-destilled water. The number of associated Salmonella (adhering and invading) was determined by serial dilutions on 0.1% w/v tryptone followed by colony counts on nutrient agar. Salmonella invasion was determined by counting only bacteria located in the Caco-2/TC-7 cells. For this purpose, the monolayer incubated with Salmonella as previously described, were treated with 0.5 mL/well of gentamicin (100 μg/mL PBS) for 1 h at 37°C. Subsequently, cells were lysed and colony counts performed as described above.
Salmonella enteritidis serovar enteritidis CIDCA 101, provided by Dr. H. Lopardo, was grown in nutritive broth (Biokar Diagnostics, Beauvais, France) for 18 h at 37°C (Golowczyc et al., 2007 (link)). Confluent Caco-2/TC-7 monolayers were washed twice with sterile PBS (pH 7.2). Cells were pre incubated for 1 h at 37°C in a 5% CO2—95% air atmosphere with 250 μL EPS solutions (300, 500 and 800 mg/L in DMEM) or 250 μL DMEM in the case of Salmonella association and invasion controls. Afterwards, 250 μL of Salmonella suspension (1 × 107 CFU/mL) were added to each well and incubated 1 h at 37°C in a 5% CO2—95% air atmosphere. For association assays, cells were washed three times with PBS and lysed with 500 μL/well of bi-destilled water. The number of associated Salmonella (adhering and invading) was determined by serial dilutions on 0.1% w/v tryptone followed by colony counts on nutrient agar. Salmonella invasion was determined by counting only bacteria located in the Caco-2/TC-7 cells. For this purpose, the monolayer incubated with Salmonella as previously described, were treated with 0.5 mL/well of gentamicin (100 μg/mL PBS) for 1 h at 37°C. Subsequently, cells were lysed and colony counts performed as described above.
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