Isolation and Culture of Astroglial and Neuroglial Cells

Astroglial cell cultures were isolated from the brains of 1–2-day-old mouse according to the modified McCarthy and de Vellis protocol [87 (link)]. The brains were extracted, the cerebral cortex was separated, the meninges were removed, and the tissue was minced and incubated in 0.05% trypsin-EDTA solution at 37 °C for 30 min. After enzymatic digestion, the tissues were washed twice in PBS and then dissociated by glass Pasteur pipette in a culture medium consisting of DMEM (PanEco, Moscow, Russia), 1 g/L D-glucose, and 10% FBS (Biosera, Kansas City, MO, USA), with the addition of 2 mM glutamine (PanEco, Russia). The suspension of cells was transferred on ventilated culture vials (Costar, Washington, DC, USA) precoated with poly-D-lysine (10 μg/mL). The cells were cultivated at 37 °C and 5% CO₂. After 5–6 days, the cultures were shaken on an orbital shaker at 200 rpm for 16 h to detach and remove microglia. After 10 to 20 days, in vitro astrocytes were used for experiments.
Neuroglial cortical cultures were prepared in a similar manner as astroglial cultures, but cells were cultured in Neurobasal-A medium containing glutamine, B-27 (2%, Thermo Fisher Scientific, Waltham, MA, USA, RRID: CVCL_A315) and gentamicin (20 μg/mL, Sigma-Aldrich, St. Louis, MI, USA, Cat #G1397) [25 (link)].