5 fluorodexoyuridine fudr

Cortical neurons were harvested from rat embryos euthanized (we have complied with all relevant ethical regulations), at embryonic day 18 and plated in 24-well plates as previously described [40 (link)], but without glass cover slips. At DIV4, 300 μL media was removed from each well and replaced with 500 μL complete neurobasal media (neurobasal (Gibco) supplemented with 2% (v/v) B27 supplement (Life Technologies), 1% (v/v) Glutamax (Life Technologies), 1% (v/v) penicillin-streptomycin (VWR, 5 units/mL of penicillin and 5μg/mL streptomycin), and 10 μM (C_f) 5-fluorodexoyuridine (FUDR, Sigma-Aldrich) to inhibit glial cell growth. Subsequently, approximately 30% of the media in each well was replaced with fresh complete neurobasal media every 3 days. Neurons were maintained at 37 °C under 5% CO2.

All protocols were approved by Stanford University’s Institutional Animal Care and Use Committee. Cortical neurons were harvested from male and female rat embryos at post embryonic day 18 following euthanasia by CO₂ asphyxiation, as previously described (Loh et al., 2016 (link)), and described again below. Dissected cortical tissue was digested in papain (Worthington) and DNase I (Roche) for 30 min at 37°C. The cell suspension was filtered through a 40 μm nylon cell strainer. Cells were cultured in 24-well plates at 37°C, 5% CO₂ either on a glass coverslip (0.1 mm thick) or directly on the plate. Neurons were grown in complete neurobasal media (Gibco) supplemented with 2% (v/v) B27 (Life Technologies, 1% (v/v) Glutamax (Life Technologies), 1% penicillin-streptomycin (VWR, 5 units/mL penicillin, 5 ug/mL streptomycin). At DIV4, 300 μL media was removed from each well and replaced with 500 μL complete neurobasal media supplemented with 10 μM 5-fluorodexoyuridine (FUDR, Sigma-Aldrich) to inhibit glial cell growth. Subsequently, approximately 30% of the media in each well was replaced with fresh complete neurobasal media every 3 days.