Lucifer yellow dye

To analyze OE-MSC transmigration, we used a model of BBB with two compartments separated by a porous membrane (pore size: 1 μm), already described by Molino et al. [32 (link)]. Briefly, rat endothelial cells from blood cerebral microvessels were cultured in the top compartment on polyethylene insert filters coated with collagen IV and fibronectin, at a cell density of 500,000 per insert in 6-well culture plates. Rat glial cells were cultured in the bottom compartment at a density of 16,000 cells/cm². For both compartments, the culture medium was composed of DMEM/Ham F12 supplemented with 20% bovine serum depleted in platelets, 2 ng/mL fibroblast growth factor 2 (FGF2), and 500 nM hydrocortisone [32 (link)]. For each experiment, 3 to 4 conditions were evaluated in duplicate: 0, 120,000, and 500,000 OE-MSCs were seeded on top of the endothelial cells, then incubated for 24 h. Permeability to the Lucifer yellow dye (LY, Sigma-Aldrich L0259) was next measured over 60 min using fluorimetry, as described [32 (link)].

Intercellular communication via gap junctions was characterized using dye transfer after scrape loading, as described before [28 (link)]. In brief, confluent HUVEC monolayers in six-well plates were used. Three cuts were applied with a surgical blade in each well after washing with PBS containing Ca²⁺ and Mg²⁺ three times. Thereafter, cells were loaded with lucifer yellow dye (Sigma-Aldrich, Darmstadt, Germany) by incubation of the cells in a warmed dye solution (1 mg/mL) at room temperature for 5 min in the dark. After this period, the dye solution was rigorously washed away and the HUVECs were fixed using 10% formalin. Fluorescence and bright field images of the individual scratches were immediately acquired thereafter using the motorized inverted fluorescence microscope described above. Images were taken using a 10× objective and exposure times of 1/7 or 1/1900 s and stored on a hard disk at a resolution of 1360 × 1024 pixels for analysis later. The area covered by stained cells was determined as the readout of dye diffusion through gap junctions using ImageJ. Hitherto, the images were converted to black-and-white using a threshold function and the area was measured after calibration.

Lucifer yellow dye (1 mg/mL; Sigma-Aldrich, USA) was topically applied on the epidermis for 1 h. Excess Lucifer yellow was washed away with 1 × PBS, and the skin tissues were cryofrozen with OCT tissue freezing medium (Leica, USA). Tissues were cryosectioned at 8 μm and mounted with Hoechst 33,342 dye (Life Technologies, USA). Images were taken using the LSM 710 confocal microscope with the Zeiss EC Plan-NEOFLUAR 20×/0.5 NA objective. Analyses were performed with ZEN 2012 Light Edition software (Carl Zeiss, Germany).