Ultraglutammine media

The rat adrenal pheochromocytoma cell line, PC-12, was purchased from the American-type culture collection (CRL-1721, ATCC, Rockville, MD, USA), cultured in RPMI 1640 with UltraGlutammine media (LONZA, Basel, Switzerland), and supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS), and 1% penicillin/streptomycin (P/S) on BD-Biocoat™ collagen-coated plates (BD Biosciences, Oxford, UK). Cells were incubated in an atmosphere containing 5% CO₂ at 37°C.

PC-12 cells were seeded at 8 × 10⁵ cells/600 mm² into a 100-mm BD-Biocoat dish (Corning, NY, USA) and grown overnight in Roswell Park Memorial Institute (RPMI) 1640 with UltraGlutammine media (LONZA) supplemented with 10% FBS, 5% HS, and 1% P/S. After 24 h, cells were treated with 100 ng/mL NGF. On day 3, cells were treated for 2 h with 1, 3, and 12 µg/mL of anti-Nogo-A. Following this treatment, cells were treated with 1000 ng/mL of the recombinant Nogo-A peptide.
Total RNA extraction was performed using TRIzol^® (Life Technologies, Carlsbad, CA, USA). RNA retro-transcription was performed using SuperScript^® II Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA). Neuron growth-associated protein 43 (GAP43) and neurofilament light (NFL) gene expression analysis was performed using StepOnePlus™ Real-Time PCR (Applied Biosystems^®, Foster City, CA, USA) and TaqMan^® Fast Advanced Master Mix (Life Technologies). The rat probes used for this analysis were Rn01474579_m1 (GAP43) and Rn00582365_m1 (NFL; Life Technologies); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as expression housekeeping control (Rn01775763_g1; Life Technologies).