Strains at log phase was used in the expriments. For cytotoxic assay, Caco-2 cells (1 × 104)54 (link) in 96 well plate was used to detect cytotoxicity of the strains (2 × 105 CFU) with lactate dehydrogenase kit (Beyotime, Beijing, China). The percentage of cytotoxicity was calculated referring to the protocol of the kit: cytotoxicity (%) = (LDH release from infected cells –spontaneous release of LDH from uninfected cells) / (maximum LDH release from cell lysate–spontaneous release of LDH from uninfected cells) × 100%.
For cell adhesion and invasion, single layer Caco-2 cells in the 24 well culture plate were inoculated with 500 μL bacterial suspension (1 × 106 CFU) for 3 h. After washing to remove unadhesive strain, cells were then treated by trypsin digestion for 2 h. In invasion, extracellular bacteria were treated with gentamicin (100 μg/ml) and penicillin G (5 μg/ml) before trypsin treatment. The digested cells were lysed using 1% saponin and the lysis was inoculated on THB plate. The rate of adhesion (Ra) and invasion (Ri) was expressed as (CFU determined from plate / CFU original inoculum) × 100%. The relative invasion rate was expressed as Ri strain / Ri P1/7 × 100%.
For cell adhesion and invasion, single layer Caco-2 cells in the 24 well culture plate were inoculated with 500 μL bacterial suspension (1 × 106 CFU) for 3 h. After washing to remove unadhesive strain, cells were then treated by trypsin digestion for 2 h. In invasion, extracellular bacteria were treated with gentamicin (100 μg/ml) and penicillin G (5 μg/ml) before trypsin treatment. The digested cells were lysed using 1% saponin and the lysis was inoculated on THB plate. The rate of adhesion (Ra) and invasion (Ri) was expressed as (CFU determined from plate / CFU original inoculum) × 100%. The relative invasion rate was expressed as Ri strain / Ri P1/7 × 100%.
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