Ussing Chamber Assay for Intestinal Permeability

Intestinal permeability was determined using Ussing chamber analysis. The colonic mucosa was intactly scraped from the distal colon specimens, installed in a slider with a 0.3 cm² rectangular hole in the center, fixed in the U-shaped chamber, and immersed in oxygen-containing Krebs’ solution on both the serosal and mucosal sides. Then, the chamber was mounted on Ussing Chamber System (World Precision Instruments, USA). The transepithelial resistance (TER) of the colonic mucosa was recorded by an automatic voltage clamp model after a 20 min equilibration. In addition, mucosal-to-serosal permeability was assessed by fluorescein isothiocyanate conjugated dextran (FD4, FITC-dextran, molecular weight: 4 kD, Sigma-Aldrich, Madrid, Spain). After the TER recording, 1 mg/ml FD4 was added to the mucosal side of the chamber, and the same volume of Krebs’ solution was added to the serosal side without light. One hundred microliters of solution was sampled from the serosal side every 30 min over a 2 h period, and the fluorescence intensity was detected by a fluorescence spectrophotometer (485 nm/528 nm, Ex/Em, BioTek, Winooski, VT, USA). The FD4 concentration in the serosal side was evaluated by a standard curve of continuous dilutions of FD4 in Krebs’ solution.