Astrocytes were purified by immunopanning from postnatal day 5 rats or mice (see above) forebrains and cultured as previously described18. Briefly, cortices were enzymatically (papain) then mechanically dissociated to generate a single cell suspension that was incubated on successive negative immunopanning plates to remove microglia, endothelial cells, and oligodendrocyte lineage cells before positively selecting for astrocytes with an Itgb5-coated panning plate. Isolated astrocytes were cultured in a defined, serum-free base media containing 50% neurobasal, 50% DMEM, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 292 μg/ml L-glutamine, 1× SATO and 5 μg/ml of N-acetyl cysteine. This media was supplemented with the astrocyte-required survival factor HBEGF (Peprotech, 100–47) at 5ng/ml as previously descried18. A similar immunopanning protocol was used for other central nervous system cell types, with positive selection using THY1 (cortical neurons), 192 hybridoma clone (embryonic spinal motor neurons35), CD31 (endothelial cells36), O4 (oligodendrocyte lineage cells), PDGFRβ (pericytes37), CD45 (microglia/macrophages). A1 reactive astrocytes were generated in vitro by growing purified astrocytes for 6 days and then treating for 24 h with Il-1α (3 ng/ml, Sigma, I3901), TNFα (30 ng/ml, Cell Signaling Technology, 8902SF), and C1q (400 ng/ml, MyBioSource, MBS143105).
