Neuron-Astrocyte Co-Culture Transfection

Neurons and astrocytes were derived from cortices of wild-type Sprague Dawley rats at 0–1 days postnatal. Neurons were grown on a confluent monolayer of astrocytes at 5% CO₂. Astrocytes were plated on 18 mm coverslips made of German glass (Carolina Scientific) that were acid cleaned then coated with a mixture of collagen and poly-D-lysine and grown in MEM without phenol red (Gibco), supplemented with N2 (Gibco), 10% fetal calf serum (Gibco or HyClone), 20% glucose, glutamax (Gibco), and Primocin (Invivogen). When astrocytes formed a confluent monolayer, neurons were plated on top of the astrocytes. Co-cultures were maintained in neuronal medium: Neurobasal-A (Gibco) with B27 (Gibco) and without antibiotics or phenol red, as previously described (Bury and Sabo, 2011 (link), 2014 (link); Sceniak et al., 2012 (link)). Half of the medium was replaced with fresh, equilibrated neuronal medium every 3–5 days.
Neurons were co-transfected with pEGFP-GluN2B constructs (2.6 μg DNA/coverslip) along with tdTomato (0.4 μg DNA/coverslip) at 2 DIV using the calcium phosphate method (Berry et al., 2012 (link); Sceniak et al., 2019 (link)). pEGFP-GluN2B constructs were previously described (Sceniak et al., 2019 (link)). EGFP is inserted in the amino-terminal extracellular domain after the signal peptide. tdTomato-N1 was Addgene plasmid 54642.