Mouse recombinant chi3l1 protein

Primary neuronal cultures were prepared from E16 cortices of C57BL6/J mouse embryos. Briefly, embryos were decapitated and brains were dissected out. Meninges, choroid plexus and hippocampus were removed and cortices were mechanically and enzymatically disrupted in the presence of trypsin and DNase I (Sigma) for 10 min at 37 °C. Cell suspensions were filtered through a 70-µm cell strainer and plated on coverslips precoated with 0.1 mg/ml poly-D-lysine (Sigma) in 24-well plates. Neurons were seeded at a density of 75,000 cells/cm² in Advanced DMEM (Gibco) supplemented with 10% Fetal Bovine Serum (FBS, heat inactivated, Sigma), 20 U/ml penicillin and 20 µg/ml streptomycin (Gibco). Three hours after seeding, medium was replaced with serum-free Neurobasal medium (Gibco) containing 2% B27 supplement (Gibco), 1% GlutaMAX (100×, Gibco), 20 U/ml penicillin and 20 µg/ml streptomycin (Gibco). Culture medium was partially changed once per week with fresh supplemented Neurobasal medium. Cultures were maintained at 37 °C, in a humidified atmosphere containing 5% CO₂ and treated with vehicle (PBS) or diverse concentrations of mouse recombinant CHI3L1 protein (R&D Systems) for 24 and 48 hours at 11 days in vitro (DIV). Glutamate (Sigma) at 200 µM was used as a positive control of neuronal excitotoxicity.

Splenocyte, astrocyte and mixed glial cultures were untreated or treated with vehicle (PBS) or diverse concentrations of mouse recombinant CHI3L1 protein (R&D Systems) for 6, 24 and 48 hours. The effect of CHI3L1 on cell death was assessed using Fixable Viability Stain 510 (BD Biosciences) and flourochrome-labeled anti-CD45 mAb (#561869, BD Biosciences) for splenocytes or Fixable Viability Stain 510 (BD Biosciences) and anti-CD11b mAb (#25-0112-81, eBioscience) for microglia. No lineage-specific marker was used for astrocyte analysis due to the purity of astrocyte culture. Samples were acquired with in a CytoFLEX (Beckman Coulter) flow cytometer and data analysis was performed with CytExpert 2.3 software (CytoFLEX/software">https://www.beckman.es/flow-cytometry/instruments/CytoFLEX/software).