N6 2 o dibutyryladenosine 3 5 cyclic monophosphate dbcamp

Cortices from rats or mouse pups were collected at postnatal day 2 and mechanically dissociated. Meninges were carefully removed, and astrocytes were separated from other cell types using 30 and 60% Percoll gradient (GE Healthcare) and seeded into coated flasks. Cells were left to proliferate at 37°C in a humidified atmosphere containing 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM-GlutaMAX, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (VWR), 100 μg/mL penicillin-streptomycin (Thermo Fisher Scientific), 2.5 μg/mL fungizone (Thermo Fisher Scientific), 50 μg/mL L-proline (Thermo Fisher Scientific), and 50 μM of b-mercaptoethanol (Gibco). The medium was renewed on day 7, and on day 14, astrocytes were collected by trypsinization (Trypsin-EDTA, Thermo Fisher Scientific) and seeded at 70,000 cells per well in six-well plates for Western blotting and into poly-L-lysine coated 24-well plates at 35,000 cells per well for uptake measurement. On day 16, serum concentration was reduced to 3% as culture medium was renewed. When indicated, a supplement of 250 μM N⁶,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (dBcAMP) (Sigma-Aldrich) was added on day 16. For all the experiments, the astrocytes were used after 23 days in culture.