Complete neurobasal medium

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.

The established differentiated human GB cell lines U87 and U373 were purchased from the American Type Culture Collection (USA), and the T98 cell line was obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK). All of the cell lines were grown in high-glucose Dulbecco’s modified Eagle medium (DMEM; GE Healthcare, IL, USA), supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, and 100 µg streptomycin, as described in Kološa [37 (link)] and Breznik [38 (link)]. The GSCs NCH644 and NCH421k were first obtained as a gift from Prof. Christel Herold-Mende (University of Heidelberg, Heidelberg, Germany) and by Cell Lines Service (GmbH, Berlin, Germany). These were grown as spheroid suspensions in complete neurobasal medium (Invitrogen, Life Technologies, Carlsbad, CA, USA), 20 ng/mL bFGF (beta fibroblast growth factor), and EGF (epidermal growth factor) (both from Invitrogen, Life Technologies, Carlsbad, CA, USA). All of the cell lines were maintained at 37 °C with 5% CO₂ and 95% humidity and were tested for mycoplasma contamination using a MycoAlert Mycoplasma Detection Kit (Lonza Pharma & Biotech Ltd., Bend, OR, USA).

Primary SC neurons were cultured using E12.5 mouse embryos of either sex as previously described (Zahavi et al., 2015 (link)). Briefly, SCs were excised, trypsinized, and triturated. Supernatant was collected and centrifuged through a 4% BSA cushion. The pellet was resuspended and centrifuged through an OptiPrep gradient (10.4% OptiPrep, Sigma-Aldrich; 10 mm Tricine, 4% glucose) for 20 min at 760 × g with the brake turned off. Cells were collected from the interface, washed once in complete medium, and then plated in coated growth chambers. Cells were maintained in Complete Neurobasal Medium (Invitrogen) containing B27 (Invitrogen), 10% (v/v) horse serum (Biological Industries), 25 nm β-mercaptoethanol, 1% penicillin-streptomycin (PS; Biological Industries), and 1% GlutaMAX (Invitrogen) supplemented with 1 ng/ml GDNF, 0.5 ng/ml CNTF, and 1 ng/ml BDNF (Alomone Labs). Before plating, the growth plates were coated with 1.5 g/ml poly-dl-ornithine (Sigma-Aldrich) overnight at 37°C and 3 g/ml laminin (Sigma-Aldrich) for 2 h at 37°C. For immunofluorescence staining, 30,000 cells were plated on cover slides in 24-well plates. Cells were grown at 37°C in 5% CO₂.

To clarify the role of miR‐30b in cultured DRG neurons, the neurons were transfected with miR‐30b agomir/antagomir or negative control (100 pmol miR‐30b agomir: 5′‐UGUAAACAUCCUACACUCAGCU‐3′ and 3′‐CUGAGUGUAGGAUGUUUACAUU‐5′; 100 pmol miR‐30b antagomir: 5′‐AGCUGAGUGUAGGAUGUUUACA‐3′; 100 pmol negative control: 5′‐UUCUCCGAACGUGUCACGUTT‐3′ and 3′‐ACGUGACACGUUCGGAGAATT‐5′) and sema3A siRNA (5′‐GCAAUGGAGCUUUCUACUA‐dTdT‐3′) (GenePharma, Shanghai, China) with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). 24 hours after transfection, the culture medium was removed and fresh complete Neurobasal Medium (Invitrogen, Carlsbad, USA) was added. Then, the DRG neurons were harvested for RT‐qPCR, Western blotting and immunocytofluorescence 48 hours after transfection.