Fitc dextran

Permeability of gut epithelium was determined as described (24 (link)). In brief, mice fasted for 4 h were orally administrated with 60 mg/100 g body weight permeability tracer of fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight, 4,000) (Sigma, St. Louis, MO, USA) and peripheral blood was collected 4 h later. Serum samples were diluted in equal volumes of PBS, and the serum FITC-dextran concentrations were determined using a fluorescence spectrophotometer (GloMax-Multi Detection System) (Promega, Madison, WI, USA) at an excitation wavelength of 490 nm and an emission wavelength of 540 nm.

In vivo intestinal permeability assay to assess barrier function was performed using Fluorescein isothiocyanate conjugated dextran (FITC-dextran, Sigma-Aldrich) method. For permeability of the small intestine, mice were water starved overnight then orally gavaged with FITC-dextran (40 mg/100 g body weight). For colonic permeability, FITC-dextran (20 mg/100 g body weight) were rectally administered twice with a 2-h break. After 4 h (for colonic permeability, 4 h after initial administration), mice were anesthetized and blood was collected and saved in the dark. Serum was then prepared from the blood, diluted 1:1 with PBS, and measured in a 96-well microplate in duplicates for the concentration of FITC in the serum by Glomax (Promega) with an excitation wavelength of 485 nm and an emission wavelength of 528 nm, using serially diluted FITC-dextran as the standards. Colonic histopathology was graded on a scale of 0 to 4 for each of the following six categories: inflammation, edema, epithelial defects, crypt atrophy, hyperplasia, and dysplasia^{62 (link)}. Only inflammatory histopathology was observed in our samples. All slides were scored in a blinded fashion (Eden).