Fluorescein isothiocyanate dextran

SCE cells were prepared by the same method used for TEER measurements as described above. SCE cell monolayers were then stimulated with 1, 5, or 25 μmol/L of K-115, Y-27632, or fasudil. A tracer, fluorescein isothiocyanate (FITC)-dextran (average molecular weight, 4000; Sigma-Aldrich), was simultaneously applied at 50 μmol/L to culture well basal compartments. Culture medium was collected from the apical side for fluorescence measurements at 60 min after the addition of tracer with an equal volume of the culturing medium added to replace the removed medium. FITC-dextran concentrations in collected medium were measured using a multimode plate reader (Gemini XPS; Molecular Devices, LLC, Sunnyvale, CA) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm. The fluorescence intensity of normal medium was measured and used as the background concentration in each experiment.

IA^s/PLP 139-151 and IA^s/Theiler's murine encephalomyelitis virus (TMEV) viral capsid protein₂ 70-86, IA^b/MOG 35-55, and IA^k/Myhc-α 334-352 and IA^k/Bovine ribonuclease (RNase) 43-56 tetramers were generated as previously described [5 (link),11 (link),25 (link),28 ]. While we generated the IA^k constructs [11 (link),29 (link)], the IA^s and IA^b constructs were kindly gifted by Dr. Vijay Kuchroo, Harvard University, Boston, MA. Briefly, α and β constructs for each IA allele containing the sequences of the respective peptides were expressed in a Baculovirus system using Sf9 insect cells (Invitrogen, Carlsbad, CA) and soluble MHC molecules were obtained [5 (link),11 (link)]. IA^s, IA^b and IA^k monomers were purified on antibody columns prepared using MKS4, M5114, and 10-2.16 (Bio × Cell, West Lebanon, NH), respectively and the protein yield generally ranged from 0.5 mg to 1 mg/L [5 (link),11 (link),25 (link),28 ]. After concentrating, the soluble MHC proteins were biotinylated using biotin protein ligase at an optimized concentration of 25 μg/10 nmol of substrate as recommended by the manufacturer (Avidity, Denver, CO). The biotinylated proteins were then incubated with SA conjugated with a fluorescent dye - fluorescein isothiocyanate (FITC), phycoerythrin (PE) or allophycocyanin (APC) - at a 4:1 ratio for one hour on ice. The reagents thus prepared are referred to as tetramers. To prepare dextramers, biotinylated soluble monomers of all three IA alleles (IA^s, IA^b and IA^k) containing the peptides were coupled to activated dextran backbones (kindly provided by Immudex Aps, Copenhagen, Denmark) at various molar ratios in 1 × Tris Hcl 0.05 M, pH 7.2 for 30 minutes at room temperature (RT) and the preparation of fluorochrome-labeled dextran backbone has been previously described [16 (link)]. Fluorochrome-labeled dextramers were prepared by mixing biotinylated IA monomers with dextran molecules. For example, dextramer-PE reagents were prepared by mixing 1.6 × 10^-11 moles of dextran-PE molecules with 3.17 × 10^-10 moles of IA^s monomers which resulted a molar ratio of 1:20. All the reagents were aliquoted and stored at -80°C or 4°C until further use.

The rats were anesthetized and fluorescein isothiocyanate-Dextran (FITC-D) (Sigma-Aldrich Corp, USA) was injected into the tail vein. Retinas and eyecups were isolated and tiled on glass slides, and fluorescence images were captured with a fluorescence microscope (Nikon). The specific operation method is carried out according to the previously reported scheme [20 (link)].

On Day 0, 12-well Transwell inserts were coated with 10 μg/ml human recombinant laminin 511-E8 in sterile PBS or 10 μg/ml BSA control coating, and left to dry for 1 h at 37 °C. After coating, H69 cholangiocytes were seeded in the apical compartment at 100,000 cells per insert. On Day 4, PBMCs were isolated from healthy volunteers, and T lymphocytes were activated with PMA and ionomycin, after which 200,000 cells were added to the basolateral compartment. The apical compartment was refreshed with H69 culture medium and the basolateral compartment contained supplemented IMDM with or without activated T lymphocytes. Co-culture was left overnight. On Day 5, 4 kDa fluorescein isothiocyanate (FITC)-Dextran permeability assays were performed. For this, medium was refreshed with 800 μl DMEM supplemented with 10% FBS and 37.5 U/ml (1%) penicillin, 37.5 μg/ml (1%) streptomycin on the basolateral side, and 250 μl of supplemented DMEM containing 1 mg/ml 4 kDa FITC–Dextran on the apical side. At timepoint 0, 100 μl of medium was transferred to a black 96-well plate to determine potential background fluorescence. In addition, an empty unseeded Transwell insert was included to determine maximal permeability of the insert itself. At t = 60, 120, 180, and 240 min, 100 μl of basolateral medium was collected per experimental condition and transferred to the black 96-well plate. FITC–Dextran fluorescence (excitation 490 nm, emission 520 nm) was measured using the CLARIOstar apparatus.