Caco-2 Cell Adhesion Assay for Bacterial Strains

The Caco-2 cell line was purchased from the Institute of Biochemistry and Cell Biology (SIBS, CAS, China). Caco-2 cell culture was performed using the method reported by Fernández et al. [32 (link)], with modifications. Briefly, Caco-2 cells were inoculated into Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and 1% (v/v) antibiotics (100 U/mL penicillin sodium, 1.0 μg/mL streptomycin) and incubated at 37°C (5% CO₂). The culture medium was replaced the next day. After 15–18 days, the Caco-2 cells were transferred into six-well plates and grown to confluence. A monolayer of Caco-2 cells (5 × 10⁵ CFU/mL/cm²) was used for adhesion assays after washing twice with PBS (pH 7.2).
The wild-type and mutant strains of KLDS1.0391 were routinely grown overnight in MRS broth. Then, the cells were collected (8000 ×g, 4°C, 10 min), washed twice with PBS (pH 7.2), and resuspended in DMEM to the final concentration of 10⁸ CFU/mL. The suspensions (5 mL) were added to the abovementioned wells containing Caco-2 cells and incubated for 2 h (37°C, 5% CO₂). At the end of the incubation period, the Caco-2 cell cultures were washed twice with prewarmed PBS (37°C, pH 7.2) to remove the nonadherent cells and then treated with Triton X100 (1%, 10 min) to release the adhered bacterial cells. The adhesion ratio of bacterial cells was calculated by comparing the viable count on MRS agar plates before and after adhesion.