Poly d lysine pdl

Hippocampal glio-neuronal cell cultures were obtained from postnatal (P0–P2) C57BL6J (WT) mice and NOX4^−/− mice. Mice brains were quickly removed, and hippocampal tissue was isolated and transferred into ice-cold HBSS (Gibco). The tissue was treated with 5 ml trypsin–EDTA (0.25%; Sigma-Aldrich) for 10 min at 37 °C to facilitate cell dissociation. Residual trypsin was removed and the tissue was triturated with fire-polished Pasteur pipettes, plated on poly-d-lysine (PDL, Sigma-Aldrich) pre-coated coverslips (CS) and cultured in neurobasal medium (Gibco) supplemented with B-27 (Gibco), 0.5 mM GlutaMAX (Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich) at a density of 150,000 cells. Cultures were kept at 37 °C in a humidified atmosphere containing 5% CO₂ for 12 to 20 days before experiments.

Choosing the cerebral cortex of mice (1-3 days old) to extract primary astrocytes, the cortex was digested by 0.125% trypsin-EDTA (Gibco, Grand Island, NY, USA), then centrifuged and incubated in DMEM/F12 complete medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 9-11 days, the cells were shaken at 260 rpm at 37° C for 16 h to purify. Finally, purified astrocytes were obtained.
Choosing the cortex of fetal mice (16–18 days old) to isolate primary neurons, the culture flasks were pretreated with poly-d-lysine (PDL) (Sigma-Aldrich, St. Louis, MO, USA). The fragment was digested with 0.125% trypsin and grown with DMEM (Gibco, Grand Island, NY, USA) for 5 h, then replaced with neurobasal medium containing 2% B27 (Gibco, Grand Island, NY, USA) and 0.5 mmol/L glutamine (Sigma-Aldrich, St. Louis, MO, USA).
Choosing Adult Brain Dissociation Kit and ACSA-2 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for magnetic isolation of astrocytes from adult mice. The former was used to digest the cortex. Then, purified astrocytes were obtained. Astrocytes were incubated for 15 min at 4° C with ACSA-2 MicroBeads and separated from single-cell suspension in a magnetic field using MS columns, MACS MultiStand and QuadroMACS (Miltenyi Biotec, Bergisch Gladbach, Germany).

hES cells were cultured in low attachment 96-well plates for 4 d to generate embryoid bodies (EBs). EBs were then transferred to Matrigel-coated six-well plates for attachment and further cultured in neural induction medium (STEMdiff, STEMCELL Technologies) for 4–5 d. Next, cells were plated on laminin-coated (10 µg/ml, Thermo Fisher Scientific) six-well plates and cultured in human NSC medium (NeuroCult, STEMCELL Technologies) supplemented with 20 ng/ml of epidermal growth factor (EGF) and 10 ng/ml of basic fibroblast growth factor (bFGF; Peprotech) for 7 d for NPC maturation, which were then used for transplant.
For proliferation and differentiation assays, 12-mm glass coverslips were pre-coated with 50 µg/ml poly-D-lysine (PDL; Sigma Aldrich) and 10 µg/ml laminin (Thermo Fisher Scientific). NPC were seeded on coverslips at 12 000 cells/cm² density and cultured in human NSC medium (NeuroCult, STEMCELL Technologies) supplemented with 20 ng/ml of EGF and 10 ng/ml of bFGF (Peprotech). For differentiation assays, cells were cultured in the same media, but with withdrawal of mitogens (EGF, bFGF) for 5 d. Cells were fixed with 4% paraformaldehyde (pH 7.4, Acros Organics) in PBS at 4°C for 15 min and analyzed by immunofluorescence.