Culturing Primary Neurons from Humanized Mice

Primary neurons were cultured from embryonic day (E)15.5-E17.5 humanized (Hu97/18 and Hu18/18) transgenic mice (Southwell et al., 2013 (link)). Brains were removed from embryos and stored in Hibernate-E (Gibco A1247601) overnight during genotyping. The following day, cortices with attached striatal tissue were dissected and pooled by genotype (Hu18/18 or Hu97/18). Cultures were established as in Schmidt et al. (2018 (link)). Briefly, tissue was dissociated, trypsinized, and resuspended in Neurobasal complete medium [NBC; 0.02% 10× SM1, 0.01% 100× Pen/Strep, 0.0025% glutamax in Neurobasal Media (Gibco 21103-049)]. The cells were seeded onto poly-D-lysine (PDL) coated plates at a density of 250,000 cells per well in 24-well plates and 1 × 10⁶ cells per well in 6-well plates. For immunocytochemistry, coverslips were treated with hydrochloric acid overnight at room temperature (RT) with gentle shaking and washed in ethanol and phosphate-buffered saline (PBS). Coverslips (Marinefield No. 1.5, 13 mm) were then added to wells and dried before PDL coating. Cells were maintained in a 5% CO₂/humidified incubator at 37°C. Neurons were fed with 1/10 well volume NBC twice weekly.