*4.2. Isolation of Primary Astrocyte Cultures*

Primary astrocyte cultures were obtained from the cerebral cortex of newborn C57BL/6 mice (1–3 days after birth). The use of brain tissue in the early postnatal period minimizes the risk of the presence of nondifferentiated astrocytes in cultures.

Preparation and long-term culture of primary astrocyte cultures were performed in accordance with a protocol based on studies by [27] and [28] with several modifications. Surgically isolated cerebral cortices were cleared from the meninges and then mechanically dissected. To disrupt interconnections between cells, the tissue was additionally incubated with 0.25% trypsin solution (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min in a CO2 incubator (BINDER GmbH, Tuttlingen, Germany). Next, the cell suspension was washed three times in phosphate-buffered saline (PBS) and once in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/L glucose and supplemented with 0.5% L-glutamine, 1% B27 (Thermo Fisher, Waltham, MA, USA), 0.1% sodium pyruvate (Thermo Fisher, Waltham, MA, USA), and 10% fetal bovine serum (PanEco, Moscow, Russia). The suspension of dissociated cells was centrifuged at 800 rpm for 3 min. Then, the pellet was resuspended in culture medium, and, the obtained suspension was placed on coverslips pretreated with polyethylenimine solution (1 μg/mL) (Merck KGaA, Darmstadt, Germany), which provides effective cell attachment to the substrate. The initial cell density was 4500 cells/mm2. Primary astrocyte culture viability was maintained under constant conditions of 35.5 ◦C, 5% CO2, and a humidified atmosphere in a cell culture incubator for more than 30 days. Half of the medium was replaced every third day.

#### *4.3. Immunocytochemical Analysis*

To verify the cellular content, primary astrocyte cultures were subjected to immunocytochemical staining on day 21 of culture development in vitro (DIV) (Supplementary Materials 1, Supplementary Materials Figure S1). The cultures were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by incubation with a solution of 0.2% Triton X-100/PBS for effective cell permeabilization. For immunofluorescence reactions, the cultures were then incubated for 2 h in the presence of a polyclonal chicken anti-GFAP (glial fibrillary acidic protein, marker of differentiated astrocytes) primary antibody (Abcam, Cambridge, UK, 1:1000 dilution) and polyclonal goat anti βIII-tubulin (marker of differentiated neurons) primary antibody (Abcam, Cambridge, UK, 1:750 dilution). Next, the cultures were subjected to a 45-min incubation in the following secondary antibody mixture: mouse anti-chicken Alexa 647 (Thermo Fisher Scientific, Waltham, MA, USA, 1:100 dilution) and rabbit anti-mouse Alexa Fluor 555 (Thermo Fisher Scientific, Waltham, MA, USA, 1:100 dilution). The stained material was observed using a Zeiss 510 NLO fluorescence confocal microscope (Carl Zeiss, Oberkochen, Germany).
