Mw 4000

Permeability of the mouse gut was assessed using a FITC permeability assay. Food and water were removed from mouse cages for four hours. After four hours, mice were gavaged with 60 mg of FITC-dextran (MW 4000, Sigma-Aldrich, St. Louis, MO, USA) per 100 g of mouse [24 (link)]. Three hours post gavage, mice were bled into 100 μL heparin, and plasma was separated by spinning the blood at 2000 rpm for ten minutes at 4 °C. Then, 50 μL of each sample was loaded into a 96-well plate in duplicate to measure FITC concentration at emission and excitation wavelengths of 520 nm and 490 nm, respectively, using a TECAN fluorescence spectrophotometer and Magellan software. FITC-dextran standards were diluted in plasma from unmanipulated mice. Fluorescence from the negative control samples (plasma from unmanipulated mice) was subtracted from fluorescence of the standards and experimental samples.

Epithelial barrier function was assessed by in vivo permeability assay using FITC-labeled dextran according to the method described previously [17 (link)]. Briefly, after 4 h fasting mice were orally administrated with FITC-labeled dextran (44 mg/100 g body weight), (MW 4000; FD4, Sigma-Aldrich Co., St Louis, MO). Blood was collected 5 h later via cardiac puncture and was then spun at 1,000 rpm for 20 min to separate serum from whole blood cells. Fluorescence intensity in the serum was determined at 485-nm excitation and 520-nm emission wavelengths. FITC-dextran concentrations were determined using a standard curve generated by serial dilution of FITC-dextran.

For in vivo intestinal permeability studies, fluorescein-5-isothiocyanate (FITC)-conjugated dextran (MW 4000; Sigma-Aldrich, St. Louis, MO) was administered by oral gavage at a concentration of 60 mg/100 g of body weight. Blood was collected retro-orbitally two hours later, and serum was harvested. Fluorescence intensity was determined with a fluorescence spectrophotometer (BioTek) at emission and excitation wavelengths of 485 nm and 528 nm, respectively. FITC concentration was measured from standard curves generated by serial dilution of FITC-dextran 15 (link).

Briefly, at 42 d, one chick from each light treatment group was given a gavage of 1 mL fluorescein isothiocyanate dextran solution (FITC-d; 2.2 mg/mL; MW 4000; Sigma, Adrich, St. Louis, MO, USA). The chicks were euthanized 2 h later and blood was collected from their veins. After centrifugation at 1000× g for 15 min, the serum was collected. Next, the FITC-d concentration per mL of serum was determined by a microplate reader (485 nm excitation and 535 nm emission, Synergy HT; BioTek, Winooski, USA). In addition, PBS was used to dissolve FITC-d to make the standard curve and the FITC-d concentration per mL of serum was determined according to the standard curve.

In vivo permeability assay to assess intestinal barrier function was performed using an FITC-labeled dextran method. Briefly, 8- to 12-week-old mice fasted for 12 hours were rectally administered with FITC-labeled dextran (2 mg/10 g body weight, MW 4000, SIGMA). Blood was collected after 3 hours and serum fluorescence intensity was measured. FITC-dextran concentrations were calculated from standard curves generated by serial dilution of FITC-dextran.

Intestinal permeability was measured using fluorescein isothiocyanate dextran (FITC-d; MW 4000; Sigma-Aldrich, St. Louis, MO, USA), modified by the method described by a previous study [61 (link)]. Briefly, on Day 19, one bird was randomly selected from each cage and orally inoculated with 1 mL of the FITC-d solution (2.2 mg/mL). Blood was collected from the birds two hours post-inoculation and allowed to clot for two hours in the dark at room temperature. After centrifugation at 1500× g for 15 min, serum was collected. A standard curve was generated using serial dilutions of the FITC-d stock, while a dilution buffer was created using pooled serum from birds on a basal diet. Standards and samples were loaded onto black 96-well plates (Greiner BIO-ONE, Monroe, NC, USA), and the FITC-d concentrations were quantified using a spectrophotometer (VICTOR Nivo Multimode Microplate Reader, PerkinElmer, Shelton, CT, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.