Enriched Neuronal Culture Protocols

Embryonic cortical neurons were isolated by standard procedures. E16.5 embryonic cerebral cortices were treated with 0.25% Trypsin-EDTA and dissociated into single cells by gentle trituration. Cells were suspended in Neurobasal medium supplemented with B27 and 2 mM GlutaMAX, then plated on coverslips or dishes coated with poly-L-Lysine (0.05 mg/mL) diluted in boric buffer. Enrichment of neuronal culture was performed using both a previously reported method and a modified protocol. For the previously reported method [5 (link)], 5 μM 5-fluoro-2'-deoxyuridine (FdU) from Sigma (F0503, St. Louis, MO, USA) was added into the culture at DIV4. Cultures were then replenished by replacing half of the volume of medium, but still containing 5 μM FdU, every 3–4 days until DIV14. For our modified protocol, 1 or 2 μM FdU added at DIV4 and incubated for 24hr to kill the proliferating cells. Medium containing FdU was then replaced with fresh medium without FdU (old to new NB medium in 1:1 ratio). Half of the medium was then replaced every 5 days until at least 14 days in vitro (DIV) before any treatment.
Murine neuroblastoma N2a cells were cultured in standard DMEM media supplemented with 10% FBS for routine passage. N2a cells and primary cortical neurons were treated with lipopolysaccharide (LPS, 1 μg/ml) (L2880, Sigma), tumor necrosis factor alpha (TNFα, 100 ng/ml) (#1050, BioVision, Milpitas, California, USA) or interleukin 1 beta (IL1β, 50ng/ml) (#4128, BioVision) for 1 and 24 hours. Celastrol (Sigma) was added to the cultures for 24 hours to study NFκB signaling pathways. For histological study, cells were washed with PBS and fixed in 4% PFA for 15 minutes. After rinsing in PBS, cells were immersed in 0.1% PFA for long-term storage.