2.8. Bacterial Adhesion and Invasion Assay

The Caco-2 (RRID: CVCL_0025) cell lines were procured from National Centre for Cell Science (NCCS), Pune, India and passaged and preserved at –196 °C. The cell lines were revived and plated onto 12-well cell culture plates (Eppendorf, Germany) with a density of 10⁵ cells/well and incubated at 37 °C with 5% CO₂ for 24 h. A monolayer of cells showing 70–80% confluence was selected for further experiment. The plates containing cells were washed with Dulbecco’s phosphate-buffered saline (D-PBS). The pre-existing medium was removed, and then added with Dulbecco’s Modified Eagle Medium (DMEM), containing 10% fetal bovine serum (FBS) w/o antibiotics prior to bacterial infection. Bacteria cells grown under different in vitro gut conditions were used to infect Caco-2 cells with a multiplicity of infection (MOI) of 10 and incubated with 5% CO₂ in an incubator. For adhesion assay, after 30 min of incubation, the monolayers were washed twice with D-PBS and lysed using 500 μL of cold 0.1% Triton X-100. The bacteria were enumerated by plating the serially diluted aliquots and grown overnight at 37 °C [20]. For invasion assays, the monolayers incubated with bacteria for one and half hours were washed with D-PBS and incubated further for 30 min with DMEM and 10% FBS-containing gentamicin (100 µg/mL) to kill extracellular bacteria. Caco-2 monolayers were washed and lysed, and intracellular bacteria were enumerated, as mentioned above.