Fitc dextran 4 000 fd 4

Colon tissue was opened along the mesenteric border, and duplicate samples were mounted in Ussing chambers (Physiologic Instruments, San Diego, CA, USA), exposing 0.1 cm² of tissue surface area to 2.5 ml of oxygenated Krebs-glucose (10 mM) and Krebs-mannitol (10 mM) at 37 °C on the serosal and luminal sides, respectively. The paracellular pathway and transcellular pathway were measured as the flux of FITC-Dextran 4000 (FD-4, Sigma Aldrich, St. Louis, MO, USA) and horseradish peroxidase (HRP Type VI, Sigma Aldrich, St. Louis, MO, USA), respectively. FD-4 (400 µg/mL) and HRP (200µg/mL) were added to the mucosal chamber and samples were collected from the serosal chamber every 30 min for 2 h. Concentration of FD-4 was measured via fluorescence at an excitation of 485 nm and an emission of 538 nm. O-dianisidine substrate was used to detect HRP at an absorbance of 450 nm. Flux was expressed as ng of FD4 or HRP transported per cm² of membrane per hour. Tissue samples with >1.2 × 10⁴ ng/cm²/h of FD4 were considered to be damaged and excluded for both HRP and FD4. In addition, tissue replicate samples that varied by more than five-fold or which became damaged while in the Ussing chambers were also excluded. Flux is calculated as the mean of tissues replicates (where available) 60 min after the addition of FD4 and HRP.