Obtained cell clusters were isolated with a 40-μm mesh filter (Becton Dickinson) and magnetically separated into a CD4+ or into CD4+CD25high/– fractions using a Miltenyi MACS® kit according to the suppliers manual. A proportion of the CD25high T-cell population was checked for Foxp3 expression with the purity≥85% in all experiments. Peripheral blood was drawn directly from the heart of sacrificed mice. For CNS-derived lymphocyte flow cytometry, a Percoll density gradient was used as described previously 29. In brief, mice were sacrificed with CO2 and immediately perfused with 10 mL of PBS before harvesting Neratinib datasheet the brain and spinal cord. The tissue was,
similar to the lymph nodes, mechanically homogenized in PBS, layered on a 30%/50% Percoll gradient and centrifuged without brake at 600×g for 30 min. After removing the top layer of myelin, lymphocytes were harvested at the Percoll interphase. MBMEC were isolated according to Weidenfeller et al.30. The obtained capillary fragments were seeded onto CollagenIV/fibronectin-coated Vemurafenib mw membranes of transwell inserts (6.5 mm Transwell® Pore Polyester Membrane Insert, pore size 3.0 μm, Corning, 2 inserts/mouse brain). Cells were incubated in DMEM high glucose with 2 mM L-glutamine, 100 U/mL
penicillin, 100 μg/mL streptomycin (PAA), 20% plasma derived bovine serum (First Link), 10 ng/mL basic fibroblast growth factor (Peprotech), 100 ng/mL heparin and 4 μg/mL pyromycin (Sigma-Aldrich) for 3 days followed by an additional 2 days of incubation without pyromycin. At this time, the monolayer reached confluence, which was randomly monitored by TEER measurements
(confluence at TEER plateau). Freshly isolated and magnetically separated fractions of CD4+, CD4+CD25high or CD4+CD25− T cells (6×105/insert) were applied on 3.0-μm pore polyester membrane transwell inserts (Corning) with or without a MBMEC layer grown onto the microporous membrane in RPMI1640 with 100 U/mL penicillin, 100 μg/mL streptomycin (PAA) and 2% B-27 serum free supplement (Gibco). T cells from three compartments were harvested after an incubation period of 18 h. Each transwell insert was removed from the well plate; cells from the upper chamber were collected by transfer of the cell suspension into a new conical and Arachidonate 15-lipoxygenase rinsing with PBS two times to ensure removal of all remaining T cells. T cells from within the MBMEC layer were harvested by incubating the cell layer with Accutase (PAA) for 10 min at 37°C and 4% CO2. The cells were then detached by rinsing with PBS and transferred into a new conical. Cells in the lower chamber were collected and wells were subsequently rinsed with PBS twice to ensure complete removal of cells. For quantification, Calibrite beads (Becton Dickinson) were added prior to harvesting the cells. Cell number was determined by counting 1×104 reference beads with a four-color FACSCalibur flow cytometer (Becton Dickinson).