Caco-2 Cell Transport Assay with Bacteria

Caco-2 cells were seeded onto Transwell-Clear inserts (12-well clusters, 6.5-mm inserts with polyester membrane, pore diameter 0.4 μm, Corning NY) at a density of 10⁵ cells/insert. Each insert was placed on top of a well in a 24-well plate with 1 ml in the bottom and 200 μL media in the top as described previously (Anderson et al., 2010 (link)). Caco-2 cells were grown for 5 days until confluence in Minimum Essential Medium Eagle (MEM) with 20% fetal bovine serum (FBS) without antibiotic-antimycotic (Gibco, Carlsbad, CA, United States) at 37°C in a humidified 5% atmosphere. TEER measurements were performed using a Millicell Electrical resistance system (Millipore, Billerica, MA, United States). When monolayer of cells reached the confluence, Caco-2 cells were co-incubated with 200 μL of bacterial culture grown to OD600 0.3 (7 × 10⁷ CFU/mL) in MEM media. Consequently, the TEER was measured after 8 h of incubation.