Poly d lysine laminin coated 8 well chamber slides

Six- to10-week-old female Swiss Webster (Charles River Laboratories) mice were euthanized by CO₂, and TG were harvested. Neuronal cultures were established after enzymatic digest as described by Bertke et al. (56 (link)) and after purification of the resulting cell homogenates using a percoll gradient as described (57 (link)). Neurons were counted and plated on either poly-D-lysine/laminin–coated 8-well chamber slides (BD Biosciences) at a density of 3,000 neurons per well or poly-D-lysine/laminin–coated 12 mm round slides (BD Biosciences) at a density of 4,000 neurons per well. Neurons were cultured without removing the nonneuronal cells that provide important growth support; therefore, these cultures contained a mixed population of neurons, satellite glial cells, and other cell types. Cultures were maintained with complete neuronal medium, consisting of Neurobasal A medium supplemented with 2% B27 supplement, 1% PenStrep, L-glutamine (500 μM), and nerve growth factor (NGF; 50 ng/ml). Medium was replaced every 2–3 days with fresh medium. ACV (100 nM) was added to the culture medium when TGs were harvested from HSV-1–infected mice.

Dissociated neurons (20k) were plated on poly-D-lysine/laminin coated 8-well chamber slides (BD Biosciences) containing a confluent monolayer of primary cortical mouse glia (Boulting et al., 2011 (link)). For survival analysis, slides were fixed at 3 and 30 days, and cultures were stained for counting. The number of ISL-positive, TUJ1-positive motor neurons (counter blinded to cell line identity) was normalized to the number on day 3 for each line. Retigabine (1 μM) or vehicle control was added from day 15 onwards. 11 total experiments (control motor neurons) and nine total experiments (ALS motor neurons) were performed from the same four separate differentiations. ER stress experiments were performed as described in Kiskinis et al. In brief, in two separate independent biological replicates, 300 ng of RNA was used to generate cDNA, of which 2 μL and AmpliTaq Gold Polymerase (Applied Biosystems) were used for PCR amplification. The amounts of spliced and unspliced bands were quantified using Image J. Quantitative RT-PCR was performed in triplicate using the iSCRIPT kit (Biorad) for cDNA synthesis and SYBR green (Bio-Rad) labeling followed by amplification using the iCycler system (Bio-Rad).