TEER measurement

Caco-2 cells were seeded onto collagen-coated membrane inserts at a density of 1 × 10⁵ cells per insert. Inserts were placed into individual wells of a 12-well plate containing 1 mL of medium in the basolateral chamber and 250 µL in the apical chamber. The culture medium was refreshed every 2–3 days. Confluent monolayers were used for all transepithelial electrical resistance (TEER) measurements.

For treatment, Caco-2 monolayers were pre-incubated with milk-derived extracellular vesicles (mEVs) for 12 hours prior to stimulation with dextran sulfate sodium (DSS) and tumor necrosis factor-α (TNF-α). On the day of the experiment, baseline resistance was measured, after which the apical medium was replaced with DSS+TNF-α solutions. TEER was recorded every 2 hours using a voltohmmeter by gently transferring each insert into the electrode chamber with sterile tweezers.

TEER values were calculated using the following equation: TEER = (R_m − R_i) * A. R_m is transmembrane resistance, R_i is the intrinsic resistance of cell medium, and A is the surface area of the Transwell membrane.