2.4.2. Primary Microglia Cell Culture

Microglia neuronal cultures were prepared from Wistar rat puppets at day 0 to 2 (P0–P2). Animal procedures were reviewed and approved by the Local Ethical Committee for Animal Research of the University of the Basque Country (UPV/EHU, CEEA, ref. M20/2017/035). All the experiments were performed in accordance with the European Community Council Directive on "The Protection of Animals Used for Scientific Purposes" (2010/63/EU) (22 September 2010) and with Spanish Law (RD 53/2013) (8 February 2013) for the care and use of laboratory animals.

Primary microglia cell cultures were obtained following Chen et al. protocol with slight modifications [43] (Figure 1B). Puppets' brains were removed and kept on ice in a Petri dish (10 cm ∅) with HBSS under magnifying glasses for removing the meninges and selecting the brain area of interest. Then, the cortex of the brain was collected and incubated with trypsin for 15 min at 37 ◦C. The tissue was incubated with DNase for 30 s and, afterwards, the trypsin was inactivated and the tissue was washed twice with DMEM GlutMAXTM FBS 10%, P/S. Finally, the tissue was mechanically dissociated by several passages through a 5 and 2 mL pipette and, then, passed through a 70 µm nylon strainer and centrifuged for obtaining a cell pellet. The obtained pellet was resuspended in DMEM GlutMAXTM FBS 15%, P/S and incubated in a poly-l-lysine-coated flask at 37 ◦C in a humidified 5% CO<sup>2</sup> atmosphere. After 3 days, full media were changed to DMEM GlutMAXTM FBS 10%, P/S. After this, half media changes were done every 2–3 days for maintaining this glia mixture culture for 10–14 days. After that period, the flask media were removed and replaced with DMEM GlutMAXTM FBS 15% P/S 24 h before microglia cell isolation started. Microglia cells were detached from the astroglia layer by shaking the flasks at 250 rpm for 1.5–2 h. Then, primary microglia cells were removed from the flask, resuspended in complete DMEM with FBS 15% and seeded in poly-l-lysine-precoated 96-well culture plates (for cell viability and cytokine release assays) and in 24-well culture plates with glass coverslips (for the immunofluorescence assay) at a density of 50 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well and 150 <sup>×</sup> <sup>10</sup><sup>3</sup> cells/well, respectively.
