Lucifer yellow

Scrape loading and dye transfer assay was performed to detect the gap junction intercellular transference in the living cells as previously described.^{21 (link),23 (link),44 (link)} Briefly, small molecular (MW < 900) dyes (Lucifer yellow, MW457, L0259, Sigma) were introduced and their intercellular movement through gap junctions was detected by CLSM. Firstly, the adherent cells were treated with TGF-β1 as experimental group. After treatment, cells are required to grow to a full confluence (100%), and then were washed with CaMg-PBS and then three scrapes were made using a surgical blade needle in the presence of 1 mg·mL⁻¹ Lucifer yellow (L0259, Sigma). Cells were incubated for 7 min at room temperature. After rinsing thoroughly to eliminate background, dye transfer was captured using CLSM.

The 3D models were incubated for 4 hours with 1 mM Lucifer yellow (L0259, MilliporeSigma) solution on their upper surface, then PBS washed and cryoembedded. Images were captured using an Epifluorescence Leica microscope.

The changes in permeability across the in vitro monolayer were detected using Lucifer Yellow (LY) (Sigma), a fluorescent paracellular permeability marker. A stock of 1 mg/mL was prepared in 18 MΩ water and stored at 4°C until needed. The permeability study was conducted for 4 h. For the experiments, 100 μL of 50 μM LY were added to the apical chamber of Transwells along with the different samples. A sample of 100 μL was taken from the basolateral chamber every 15 min for the first hour and every 30 min for the subsequent 3 h. Each sampling was coupled with the addition of 100 μL of fresh DMEM (or low glucose DMEM for HG study) to the wells. The samples were collected in a 96-well opaque black bottom plate (Corning). At the end of the exposure experiment, the 96-well plate was read using a Synergy 2 plate reader, controlled by Biotek's Gen5™ Reader Control and Data Analysis Software. The resulting fluorescence values were converted to amount of LY (μg) using a LY standard curve.

For paracellular permeability, Caco-2 cells were plated on a transwell insert for 14 days. Lucifer Yellow (LY, Sigma) was added to the apical compartment. From the basolateral compartment, 50 μl sample was transferred in a 96-well plate, and the fluorescence was quantified using a fluorescence microtiter plate reader at 428/549 nm. For 3D permeability assay, colonic organoid or Caco-2 spheroid-like structures were incubated with 4 kDa fluorescein isothiocyanate-labeled dextran (FITC-4D, Sigma) for 24 h. The flux of FITC-4D from the basal to the luminal compartment (L/B ratio) was assessed using confocal microscopy^{19 (link)}. Confocal images were taken with Leica laser scanning confocal microscope (Leica Microsystems GmbH) and processed using ImageJ software^{61 (link)}.