Horse serum

Sprague-Dawley rats were intraperitoneally anesthetized with 50 mg/kg sodium pentobarbital. After removal of the meninges, gray matter from the cerebral cortex was cut into small pieces and a single cell suspension was obtained by mechanical trituration. Subsequently, cells were centrifuged for 5 minutes at 1,000 r/min, resuspended and seeded in astrocytic medium consisting of 89% Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco, Big Cabin, OK, USA), 5% fetal calf serum (Biowest, Val de Loire, France), 5% horse serum (Biowest), 1% penicillin/streptomycin (Biowest). Contaminating oligodendrocyte precursor cells were removed by shaking the culture flasks several times. Cells were passaged and seeded on glass coverslips at a density of 60,000 cells per coverslip in the same medium.

On the third day of proliferation, MuSCs at 90% confluency were used for myogenic differentiation. The cells were cultured in a differentiation media consisting of DMEM containing 2% (v/v) horse serum (Biowest, Nuaillé, France), 1× GlutaMAX (Gibco), 1× AA, and 1× β-mercaptoethanol (Gibco) for 2 days without media changes. After myofiber formation from the MuSCs was evident, the cells were fixed with 4% paraformaldehyde or treated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for further analysis.

Neuroscreen-1 (NS-1), a PC12 cell line subclone, was obtained from Thermo Fisher. Cells were maintained in Roswell Park Memorial Institute 1640 Medium (RPMI medium 1640, 21875, Thermo Fisher) containing 10% horse serum (Biowest, Nuaillé, France), 5% fetal bovine serum (Biowest), 1 mM sodium pyruvate (Thermo Fisher) and antibiotics (Thermo Fisher) at a temperature of 37°C in 5% CO₂. For exposure experiments, cells were seeded in collagen I-coated Falcon 6- or 96-well plates (Corning, Corning, NY, USA) at 5 × 10⁴ and 6 × 10³ cells per well respectively, and maintained in 15% serum medium for 24 h. One day before exposure, the medium was replaced with RPMI 1640 medium containing 1% horse serum and 0.5% fetal bovine serum. It was performed concomitantly with the treatment with NGF (200 ng/ml) in an exposure medium designed to keep the pH buffering in the non-gassed incubator of the exposure system. It consisted of 1.5% serum powder reconstituted RPMI 1640 medium, without NaHCO₃, with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and with antibiotics. During exposures, culture plates were sealed with AeraSeal sealing films (Excel Scientific, Victorville, CA, USA) to avoid extended medium evaporation.