Schwann cell growth supplement scgs

Surgical vestibular schwannoma (VS) and great auricular nerve (GAN) specimens were obtained from patients undergoing indicated procedures at Massachusetts General Hospital and Massachusetts Eye and Ear. The study was conducted in accordance with the Helsinki Declaration of 1975 and written informed consent was obtained from all subjects prior to inclusion. GAN (control) samples were obtained from patients undergoing benign parotidectomy unrelated to VS, during which this nerve is routinely sacrificed. Patients who had received radiation therapy prior to surgery were excluded. All VS and GAN samples were received and processed according to protocols approved by the Human Studies Committee of Massachusetts General Hospital and Massachusetts Eye and Ear (Board Reference #14–148 H). Following surgical resection, VS or GAN tissue was immediately placed in saline solution, transported to the laboratory, and cultured. Our methods describing precise protocols for surgical specimen collection, processing, and primary cell culture are published in detail^{24 (link),25} . Mouse Nf2^−/− Schwann cells used in our mouse allograft model were maintained in 10% Schwann cell medium containing Schwann cell growth supplement (SCGS, ScienCell).

Rat primary Schwann cells (pSC) were derived from neonate rat sciatic nerves and obtained along with the Schwann cell media (SCM) and Schwann cell growth supplement (SCGS), from ScienCell Research Laboratories™ (Carlsbad, CA). Palmitic acid (PA), DHA, and poly‐L lysine were provided by Sigma‐Aldrich (St. Louis, MO). Fatty acid‐free BSA and LY294002 were purchased from EMD Biosciences (San Diego, CA). MedChem Express provided our NVP‐BKM120 (Princeton, NJ). Annexin V FITC and 7‐amino‐actinomycin D (7AAD) were purchased from BD Biosciences (San Diego, CA) and eBioscience (San Diego, CA) respectively. Finally, we obtained the antibodies to total AKT (mouse, Cat# 2920, http://scicrunch.org/resolver/AB_1147620) and phosphorylated AKT Ser473 (rabbit, Cat# 4060, http://scicrunch.org/resolver/AB_2315049) or Thr308 (rabbit, Cat# 2965, http://scicrunch.org/resolver/AB_2255933), and rapamycin from Cell signaling Technology (Danver, MA), the actin (mouse, Cat# A5441, http://scicrunch.org/resolver/AB_476744) from Sigma‐Aldrich (St Louis, MO) and Torin 1 from Tocris Bioscience (Bristol, UK). All of these antibodies are commercially available see characterization details in Table 1.