Isolation and Purification of Astrocytes from Mouse CNS

Mice were housed and used in accordance with the Home Office Animal Scientific Procedures Act (ASPA), 1986 under project licence P98A03BF9. The upper cervical spinal cord-lower brainstem tissue was dissected from newborn (P0) male and female mice (n = 6 mice/each culture; C57BL/6 J, RRID: IMSR_JAX:000664 l; provided by Dr Andrea Loreto). The tissue was physically dissociated into small pieces, followed by enzymatic digestion with 0.25% trypsin and 0.2% collagenase in a mechanical dissociator (Gentle MACS Octo dissociator, Milteny Biotec) at 37 °C for 30 minutes. The obtained single cell suspension was plated in poly-D-Lysine coated T75 flasks and kept in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% antibiotic-antimycotic mix (Thermo Fisher Scientific, 15240096). At day 7, top dwelling progenitor cells and microglia were removed by overnight shaking at 100 rpm; then the culture was differentially passaged using 0.0025% trypsin to remove microglia. Finally, 0.025% trypsin was applied for 2-3 minutes to detach astrocytes for replating, while fibroblast mostly remained attached. Further purification was carried out by GLAST antibody-tagged magnetic beads by published methods^{14 (link)}, which resulted in high-purity astrocyte cultures ( > 98%).