Fitc dextran

Intestinal permeability was measured by determining the FITC-dextran concentration in the plasma of normal mice or DSS-induced colitis mice according to the procedure described in a previous study with slight modifications (Gupta et al., 2014 (link)). In brief, normal mice and colitis mice were fasted overnight. Mice were administered FITC-dextran (4 kDa; Chondrex, Redmond, WA, United States) (200 mg/kg body weight, po), while control mice were orally administered PBS (-). Four hours after oral gavage, mice were anesthetized, and blood was collected by cardiac puncture. Blood samples were centrifuged (2,500 × g, 10 minutes, 4°C), and plasma was collected. The fluorescence intensity in plasma samples was measured in black 96-well plates using a microplate reader (Tecan GENios; TECAN, Männedorf, Switzerland) at an excitation wavelength of 485 nm and emission wavelength of 535 nm. FITC-dextran concentrations were calculated from a standard curve.

An FITC-dextran/transwell assay was used to assess monolayer cell permeability, as described previously (Sedgwick et al., 2002 (link); Irwin et al., 2005 (link)). HUVECs were seeded in the upper chamber. After transfection and viral stimulation, the culture medium of the upper chamber was switched to a non-serum culture medium with 5 μg/ml FITC-dextran (Chondrex, Washington, USA, Ca #4009). A culture medium containing 5% FBS was added to the lower chamber and incubated for 1 h at 37°C. The fluorescence energy value (with an excitation wavelength of 494 nm and an emission wavelength of 520 nm) was detected by SpectraMaxM5 (Molecular Devices, USA). FITC-dextran fluorescence energy values from the upper and lower compartments were obtained; further, the permeability coefficient of dextran (Pd) in HUVECs was calculated according to the following formula: Pd (cm/s) = ([A]/t) × (I/A) × (V/[L]), where [A] is the FITC-dextran fluorescence energy value of the lower chamber, t (s) is the time interval, A (cm²) is the transwell surface area, V (m³) is the volume of the solution in the lower chamber, and [L] is the fluorescence energy value of FITC-dextran in the upper chamber. The ratio of Pd corresponds to either the si-lncRNA group or si-NC group divided by the NC group.

Mice were administered 2% DSS (160110, molecular weight 36,000–50,000; MP Biomedicals Inc., Ohio, USA; lot number Q3526) in their drinking water for 7 days. Their weight was adjusted to 16–19 g on the day of the experiment (day 0). Weight and water consumption were measured every day. Colon length was measured 7 days after starting DSS administration. The colons were fixed in 10% formalin over 24 hours and embedded in paraffin. 3.5-µm-thick sections were stained with hematoxylin and eosin (H&E). Images of hematoxylin and eosin staining were taken using BZ-X 700 (Keyence). The histological scores were analyzed according to the method previously reported^{62 (link)}.
Intestinal permeability was assessed using FITC-dextran (molecular weight; 4000, Chondrex Inc., DC, USA) according to the manufacturer’s instructions. The concentration of FITC-dextran in plasma was quantified with a fluorescent plate reader (SH-9000, Corona Electric Co., Ltd, Ibaragi, Japan).

Intestinal epithelial barrier permeability was analyzed by the oral administration of the permeability marker, fluorescein isothiocyanate-dextran (FITC-dextran), 40 kDa (Chondrex, Redmond-Woodinville, Washington, USA; Cat No. 4009). Briefly, 7- to 8-week-old mice were fasted for 4 h and then 40 kDa FITC-dextran was administered by gavage at 20 mL/kg. The mice were then allowed to remain in the cages for 1 h followed by anesthesia. Blood was withdrawn to isolate plasma. Standards were obtained by diluting the FITC-dextran stock solution in phosphate-buffered saline (PBS). Plasma was diluted in an equal volume of PBS (pH 7.4) for analysis and FITC dextran concentrations in plasma were calculated with the help of standard concentrations prepared in PBS at 0.2 µg/mL, 0.4 µg/mL, 0.8 µg/mL, 1.6 µg/mL, 3.1 µg/mL, 6.2 µg/mL, and 12.5 µg/mL. Measurement of the FITC-dextran concentration was carried out on a Cary Eclipse fluorescence spectrophotometer (Agilent, Santa Clara, CA, USA; excitation, 490 nm, emission, 520 nm). Emission signals in mice treated with 40 kDa dextran were excluded from blank values (33% normal mouse plasma in PBS).