Primary Schwann cell differentiation assay

To study gene expression in a model that estimates myelinating Schwann cell development, we employed cAMP-mediated differentiation of primary Schwann cell cultures [31]. Primary rat Schwann cells (Kerafast, Boston, MA, Catalog Number EMI010) were maintained under standard growth conditions in complete Schwann cell (SC) medium: DMEM plus 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/mL penicillin, 50 g/mL streptomycin, 25 μg/mL gentamicin, 10 nM neuregulin EGF domain, and 2 μM forskolin. Differentiation assays were completed as previously described [31]. Prior to cell plating, culture dishes were treated with 1 mL 0.01% poly-L-lysine solution (Sigma, St. Louis, MO) per 25 cm² of surface area and supplemented with sufficient volume of water to cover the bottom of the dish. Dishes were allowed to sit for 5 min at room temperature, then the solution was aspirated and the chamber was allowed to dry completely. Laminin derived from human fibroblasts (Sigma) was diluted in Hank’s Basic Saline Solution (HBSS; without calcium and magnesium) and applied to dishes at 1 μg laminin per cm². Dishes were incubated for 1 h at room temperature. The laminin solution was removed and dishes were washed with sterile water and allowed to dry completely. On day 1, primary Schwann cells were plated at 2.4 × 10⁴ cells/cm² in complete SC medium. On day 2, medium was removed and replaced with D10 medium: DMEM plus 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin, 50 g/mL streptomycin, and 25 μg/mL gentamicin. On day 3, medium was removed and replaced with D5 medium: DMEM plus 5% FBS, 2 mM L-glutamine, 50 U/mL penicillin, 50 g/mL streptomycin, and 25 μg/mL gentamicin. On day 4, medium was removed and replaced with D5 medium plus 250 μM CPT-cAMP (Axxora, Farmingdale, NY) or vehicle. Subsequently, condition-specific medium was replenished each day. On day 7, cells were washed with HBSS and incubated in 0.15% trypsin to dissociate cells from the dish. Trypsin solution was quenched with D10 medium upon cellular detachment. The resulting cell suspension was centrifuged at 200 x g for 10 min at 4 °C for downstream RNA or protein isolation.