Forebrains of P0 Mt or Mt/Ai14 pups without olfactory bulbs and meninges were collected and placed in ice-cold Ca2+- and Mg2+-free Hanks’ balanced salt solution (HBSS) supplemented with gentamicin (2 μg/ml), 25 mM d-glucose, 1 mM pyruvate, and 20 mM Hepes. Brain tissue was digested with papain (0.6 mg/ml) at 37°C for 20 min. Forebrains were washed with plating media [Neurobasal (Gibco), 5% fetal bovine serum, 4 mM GlutaMAX (Gibco), and gentamicin (2 μg/ml)]. The tissue was gently triturated and allowed to settle, and the supernatant was filtered through a 40-μm mesh-sized cell strainer. Cells were centrifuged at 420g for 4 min and resuspended in plating media. Fifteen thousand cells per well were plated with 2 × 108 viral particles/ml onto black Greiner μClear (catalog no. 781946) or Ultra-Low Base Aurora with evaporation barrier (catalog no. ABE201201B) 384-well plates (80 μl per well) precoated with poly-d-lysine (PDL) for the primary screen and rescreen, respectively. Plating and media changes were performed using an automated Biomek FXP liquid handling workstation. Four hours after plating, 75% of the plating media was replaced by feeding media [Neurobasal-A (Gibco), 4 mM GlutaMAX (Gibco), gentamicin (2 μg/ml), and 2% B27 (Gibco)]. At DIV4, 50% of the media was exchanged with fresh feeding media supplemented with 16 μM 5-fluoro-2′-deoxyuridine (FUdR) to prevent glial cell overgrowth. At DIV8 and DIV12, additional 50% media changes were made without FUdR. Cells were maintained at 37°C and 5% CO2; plates were topped with Breathe-Easy adhesive membranes (Greiner plates) to minimize evaporation.

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