Astrocyte Purification and Culture

Astrocytes were purified by immunopanning from the forebrains of P5 Sprague Dawley rats (Charles River) forebrains and cultured as previously described.^{60 (link)} In brief, cortices were enzymatically disrupted (using papain) and then mechanically dissociated to produce a single-cell suspension that was incubated on several negative immunopanning plates to remove microglia, endothelial cells and oligodendrocyte lineage cells. Positive selection for astrocytes was with an ITGB5-coated panning plate. Isolated astrocytes were cultured in a defined, serum-free base medium containing 50% neurobasal, 50% DMEM, 100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin, 1 mM sodium pyruvate, 292 μg mL⁻¹l-glutamine, 1× SATO and 5 μg mL⁻¹ of N-acetyl cysteine. This medium was supplemented with the astrocyte-required survival factor HBEGF (Peprotech, 100-47) at 5 ng mL⁻¹.^{60 (link)} Cells were plated at 5000 cells per well in 12-well plates coated with poly-d-lysine and maintained at 10% CO₂.