Fluorescein isothiocyanate labeled dextran

Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM, phosphate-buffered saline (PBS), fetal bovine serum, insulin-transferrin-selenium-X, and penicillin-streptomycin mixture were purchased from Life Technologies (Carlsbad, CA, USA). NSs (cytidine, adenosine, guanosine, and thymidine), LPS, and fluorescein isothiocyanate-labeled dextran (molecular mass of 4 kDa, FD4) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TFF3 was purchased from Cloud-Clone Corp. (Katy, TX, USA). Lipofectamine 2000 and RNAiMAX reagent were purchased from Invitrogen (Carlsbad, CA, USA). The pGL3-Basic (Promega, Madison, WI, USA) and pcDNA3.1 (+) (Thermo Fisher Scientific, Waltham, MA, USA) plasmids were used as cloning vectors. Primary antibodies specific for TFF3 (Antibodies Online), β-actin (Abcam, Cambridge, UK), ZO-1 (Thermo Scientific), occludin (MybioSouce), phospho-AKT (Cell Signaling Technology), AKT (Cell Signaling Technology), phospho-p38 (Cell Signaling Technology), p38 (Cell Signaling Technology), phospho-STAT3 (Cell Signaling Technology), STAT3 (Cell Signaling Technology), phospho-ERK1/2 (Cell Signaling Technology), and ERK1/2 (Cell Signaling Technology) were used for western blotting, immunohistochemistry, and immunofluorescence. The secondary antibodies were purchased from Abcam.

The 70 or 2000 kDa fluorescein isothiocyanate-labeled dextrans (Sigma-Aldrich) with the polydispersity of ~1.5 [40 (link)] were resuspended in phosphate-buffered saline (PBS). The fluorescent dextrans (FD) were then washed two times for 3 h through 30-kDa or 1000 kDa Vivaspin ultrafiltration spin columns (Sartorius Stedim Biotech GmbH) to remove any free fluorochromes or low molecular weight contaminants. The high molecular weight component was then resuspended in phosphate-buffered saline (PBS) at final concentration of 37.5 mg/ml and a bolus injection of 100 μl of this suspension was administered intravenously (intraorbitally) to mice in the experiments.

FRAP was performed to measure HELP hydrogel diffusivity as previously described (89 (link)). Briefly, 30 μl of gels was formed at the bottom of a clear-bottom, half-area, black 96-well plate (Greiner Bio-One). Once gels had formed, solutions of fluorescein isothiocyanate–labeled dextrans (4 mg/ml; Sigma-Aldrich) of varying molecular weights (10, 20, 40, 70, 150, and 250 k) were added to each respective well and allowed to incubate at 37°C overnight. Using a confocal microscope (Leica SPE), a 100 μm by 100 μm area of each hydrogel was photobleached using a 488-nm laser at 100% intensity for 1 min. Immediately following photobleaching, fluorescence recovery into the photobleached region was monitored for 4 min (1 frame/s) at 10% laser intensity. Images were analyzed using open source MATLAB code that was previously published (90 (link)).

To assess DC endocytic activity, BMDCs were suspended in RPMI 1640 supplemented with 10 % FCS and incubated with 1 mg/ml of FITC–dextran (Fluorescein isothiocyanate-labeled dextrans) (Mr = 40,500; Sigma Aldrich, the Netherlands) for 30 min at 4 or 37 °C. Cells were washed three times with ice-cold PBS, 0.1 % BSA and 0.01 % NaN3, and labeled on ice with appropriate mAb. The uptake was calculated as the change in mean fluorescence intensity (MFI) between cell samples incubated at 37 and 4 °C.