Caco-2 Monolayer Permeability Assay

Caco-2 cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 medium (DMEM/F-12) supplemented with 20% (v/v) fetal bovine serum and non-essential amino acids at 37 ℃ and 5% CO₂. Caco-2 cells were transferred on transwell inserts (pore size = 0.4 μm; Thermo Scientific™ Nunc™, Denmark) in 6-well plates (5 × 10⁵ cells/transwell) and cultured for 21 days to construct monolayers. The transepithelial electrical resistance (TEER) of monolayers were measured by Epithelial Volt-Ohm Meter (Millicell ERS-2, USA). The monolayers were incubated with 1 × 10⁹ CFU/mL K. quasipneumoniae or Enterococcus faecium for 1 h and with 100 mM chenodeoxycholic acid (CDCA; Sigma, Germany) for 24 h [31 (link), 32 (link)]. After incubations, the paracellular transport was measured by the clearance of FITC-dextran (4 kDa, 100 μg/mL) (Sigma, Germany) [31 (link)]. The fluorescence was measured every hour for 4 h (λexc: 493 nm; λem: 520 nm).