Cells were incubated with IFNbeta (500 U/mL) for 24h.
Growth protocol
Adult mice were sacrificed with a lethal dose of anesthetic, and the cerebrum was isolated and dissected free of the meninges. The brain was homogenized in a Dounce homogenizer, and the resulting homogenate was centrifuged at 4,000 g for 5 min. The pellet was resuspended in 18% dextran solution (molecular weight: 64,000-76,000, Sigma-Aldrich) in DMEM (Gibco) and centrifuged at 6,000 g for 10 min. After removing the supernatant and myelin debris, the pellet was resuspended in DMEM containing 1 mg/mL collagenase/dispase (Roche), 40 µg/mL DNase 1 (Roche), and 0.147 µg/mL tosyllysine chloromethyl ketone (Sigma-Aldrich), and incubated at 37°C for 75 min with occasional agitation to free endothelial cells from pericytes, perivascular macrophages, and remains of the basement membrane. The cell suspension was centrifuged at 4,000 g for 5 min, the supernatant was discarded, and cells were washed and seeded in 6-well plates coated with mouse collagen IV (BD). Cells were subsequently grown in DMEM-F12 (Gibco) supplemented with 10 % fetal bovine serum (PAA Laboratories), 2 mM L-glutamine, 100 IU/mL penicillin, 100 µg/mL streptomycin, 15 U/mL heparin (Biochrom), and 30 µg/mL of endothelial cell growth supplement (Sigma-Aldrich). 4 µg/mL puromycin (Sigma-Aldrich) was added for the first 48 h after preparation to deplete cells of non-endothelial origin.
Extracted molecule
total RNA
Extraction protocol
Cells were flushed with ice‐cold HBSS. RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer′s instructions.
Label
biotin
Label protocol
Total RNA was amplified using the Ovation PicoSL WTA System V2 and further processed using the Encore Biotin Module according to the instructions supplied by the manufacturer (NuGen).
Hybridization protocol
Biotinylated cRNA was hybridized on GeneChip® Mouse Gene 2.0 ST Arrays , that were stained, washed and scanned following standard procedures.
Scan protocol
Biotinylated cRNA was hybridized on GeneChip® Mouse Gene 2.0 ST Arrays , that were stained, washed and scanned following standard procedures.
Description
A912_04_wt-EC-IFNb4
Data processing
Background correction and normalization was performed using the RMA-Sketch implementation in Affymetrix Expression Console
