En0521

Neonatal mice (postnatal day 0–2) were sacrificed using Home Office-approved methods (proscribed by the Animals Act, 1986). Brains were dissected out and submerged in ice-cold dissection medium, consisting of DMEM:F12 (D6421, Sigma), 1% L-glutamine (A2916801, Gibco), and 1% Pen Strep (15140122, Gibco). The cortices of the brains were separated from the midbrain and cerebellum. The meninges were removed using fine forceps, and the brains were homogenized by trituration followed by digestion in trypsin (25200056, Gibco) and DNase (EN0521, Thermo Scientific) for 20 minutes at 37°C. Cells were diluted in DMEM (41965039, Gibco) with 10% FBS (F7524, Sigma), 1% Pen Strep (15070, Thermofisher) (serum medium), and centrifuged at 300 × g for 5 minutes at 10°C. Following resuspension of the pellet cells were seeded into poly-d-lysine coated imaging dishes (P35GC-0-14-C, Mattek) at a density of 5 × 10⁵ cells. The cultures were then incubated at 37°C/8% CO₂ for 16 hours to allow the cells to settle, followed by a complete media change. Cells were then cultured for 9–12 days, with a ⅔ media change (supplemented with 3 μg/mL insulin) performed every third day. Mixed glial cultures were used for imaging at 15 DIV.

RNA from tissue was isolated using the RNeasy Mini Kit (Ref:74104). Subsequently, DNAseI-treated (EN0521, Thermo Scientific) RNA was reverse-transcribed into cDNA using either Maxima Reverse Transcriptase (EP0742, Thermo Scientific) or SuperScriptVilo (11754-050, Thermo Scientific). mRNA expression levels were assessed by qRT-PCR using Fast SYBR Green Master Mix (00408995, Thermo Scientific), relative to the expression level of Gapdh or 18S (for mouse and chicken samples, respectively). For studying mRNA expression in normoxia and hypoxia conditions, mRNA levels were expressed relative to the housekeeping gene Rpl13a, due to its stable gene expression under these conditions59 (link). The qRT-PCR primers used are indicated in Supplementary Table 3.

After tamoxifen injection at P1 as described above, each mouse was administered once daily at P10-P12 with either 10U DNAse 1 (Thermo Fisher #EN0521) diluted in 0.9% NaCl or vehicle (0.9% NaCl). Treatment groups were assigned randomly. The mice were sacrificed on day P13 and used for analysis.