Millicell filter

Isolated chondrocytes from healthy, defect and OA cartilage were used either directly after isolation or after expansion to passage 2 (see Table 1 for more details). Expansion was performed in monolayers at 37°C and 5% carbon dioxide at a seeding density of 5,000 cells/cm² in expansion medium consisting of Dulbecco’s modified Eagle’s medium, 10% foetal bovine serum (Hyclone, Thermo Scientific, Etten-Leur, the Netherlands), penicillin/streptomycin (100 U/ml/100 μg/ml) and 10 ng/ml bFGF (R&D Systems, Minneapolis, MN, USA). Unexpanded chondrocytes and expanded chondrocytes were seeded on collagen type II-coated (Chicken sternal cartilage, #C9301; Sigma-Aldrich) Millicell filters (Millipore Co., Bedford, MA, USA), at 1.6 × 10⁶ cells/cm² and redifferentiated during 28 days in redifferentiation medium consisting of Dulbecco’s modified Eagle’s medium, 0.2 mM L-ascorbic acid-2-phosphate (AsAp; Sigma-Aldrich), 2% human serum albumin (Sanquin), penicillin/streptomycin (100 U/ml/100 μg/ml), 2% insulin–transferrin–selenium-X (Invitrogen) and 5 ng/ml transforming growth factor beta 2 (R&D systems).

Bovine artery endothelial cells (BAECs, NO.C-003-5C) obtained from Health Science Research Resources Bank (Osaka, Japan) were maintained at 37°C in 5% CO₂ in DMEM containing 10% FBS as described previously [18] (link). Confluent cultures were detached using trypsin/EDTA and plated on 96-well plate for evaluating cell viability, on 24-well millicell filters (Millipore, USA) for measuring endothelial permeability, and on 6-well plate for detecting NO production and mRNA expression or on 100-mm-diameter dishes for protein expression and phosphorylating signal analysis.

Brains were harvested from postnatal day 4 mice, and the hypothalamus was blocked and sectioned into 300 μm coronal slices using a manual chopper (Stoelting, Wood Dale, IL, USA). Slices containing SCN nuclei were transferred onto Millicell filters (Millipore, Billerica, MA, USA) and cultured as interface explants in a CO₂ incubator as described23 (link). After 2 days, 20 μM cytosine β-D-arabinofuranoside (ara-C, no. C6645; Sigma) was added to inhibit glial proliferation, and the media was changed every 3 days thereafter.

Transmigration of different T lymphocyte populations was assessed according to a previously described but slightly modified method [34 ]. HIBCPP were seeded on inverted Millicell® filters with 5 μm pore size (Millipore) and grown until confluency. Establishment of a proper barrier function was evaluated by TEER values above 250 Ohm/cm². Cell culture inserts were then placed in 24-well plates containing 600 µl Migration Assay Medium (MAM; DMEM with 5% FBS, 25 mM HEPES, 2% glutamine) in the presence or absence of CCL20 (MIP-3a) (500 ng/mL, Stem Cell). Human T cells were prelabelled with 1µM CellTrackerTM Green (CMFDA Dye, Life technologies) for 30 min at 37 °C before starting of the experiment. In addition, T cells were incubated with 200 nM CCR6 antagonist (CCX2553, ChemoCentryx, USA) or vehicle control (DMSO) for 20 min prior to the transmigration assay. Triplicates of 1.5 × 10⁵ T lymphocytes were added into the filter inserts in a total volume of 200 µl MAM and incubated for 8 h.
Migrated T cells were collected from the bottom compartment and counted using the Attune NxT Flow Cytometer (Thermofisher Scientific, Switzerland) by gating on CMFDA⁺ cells. The percentage of transmigrated T cells was assessed referring to the inputs as 100%.

Rat EGC/PK060399egfr (CRL-2690™) and human intestinal epithelial cells Caco-2 (HTB-37™) were obtained from the American Type Culture Collection. EGC/PK060399egfr and Caco-2 cells were grown in high glucose DMEM and MEM, respectively, supplemented with 10% FCS, 2 mM l-glutamine, and 100 U/100 μg/ml penicillin–streptomycin. Cells were incubated in a 5% CO₂ humidified incubator at 37°C. The EGC-Caco-2 co-culture system was established as described previously by our laboratory (Xiao et al., 2014 (link)). Caco-2 cells were seeded on Millicell®filters (0.4 μm pore diameter; Millipore; Billerica, MA) at a density of 5 × 10⁴ cells/cm² for up to 4–5 days. EGCs were seeded at an equal density in 6 or 24 well tissue culture plates to avoid any possible direct cell contact with Caco-2 cells. During the co-culture period, half of the culture medium in the apical and basal compartments was changed daily.

BAECs layer permeability was measured as describes previously [19] , [20] (link). Briefly, BAECs were seeded to 0.4 µm 24-well millicell filters (Millipore. USA). After incubation in high Glu with or without Res for 24 h, FITC-dextran (1 mg/ml, 40 kDa, Molecular Probes) was added to the upper compartment of the filter inserts for 3 h before determination. Mannitol is commonly used as an osmotic control. The fluorescence in the medium collected from the lower chambers was evaluated with excitation at 419 nm and emission at 528 nm.