Vegf b

Cerebral cortices from neonatal mice (1–3 days) were dissected, carefully stripped of their meninges, digested with 0.25% trypsin-EDTA and DNAse I (1 mg/ml) for 15 mins, and dispersed to single-cell level by passing through a cell strainer (70 µm). The cell suspension was then cultured at 37 °C in humidified 5% CO2, 95% air on poly-L-Lysine (Sigma) precoated 75 cm² cell culture flasks. Medium was replaced every 4–5 d. After 7–10 d cells reached confluence and astrocytes and microglia were isolated by mild trypsinization with Trypsin-EDTA (0.06%) as previously described ^{10 (link),16 (link),17 (link)}. Cells were >95% astrocytes as determined by staining with GFAP or GLAST, with less than 5% contamination of CD11b⁺ microglia cells (not shown). Conversely, microglia cultures stained CD11b⁺CD45^lowLy6C1^low >95%. After the isolation procedure, cells were further plated as required for the specific experiments. Concentrations of agents were 100 ng/ml for LPS (Sigma), 50 µg/ml for poly(I:C), 100 ng/ml for IL-1β, 50 ng/ml for TNF-α, 0.1 ng/ml TGF-α, 10 ng/ml VEGF-B (all R&D Systems), 50 µg/ml 3-Indoxyl-sulfate (Sigma), 100 nM NF-kB Blocker Bengamide B (Tocris). Unless otherwise indicated, RNA was isolated 24 hours after start of treatment. For Western Blot, cells were pretreated with I3S or vehicle for 24 h, thereafter LPS was added and protein prepared after 2 h.