Primary cell culture
Primary mixed cortical glia culture was prepared as previously described (Kinoshita, Yshii et al., 2017). Brifely, cortex from newborn C57BL/6J mice (postnatal days 1-4) was dissected in ice-cold Hanks’ balanced salt solution (HBSS) under a microscope and their meninges removed. Small pieces of cortices were incubated in a trypsin solution (GIBCO) for dissociation at 37°C for 20 minutes. DMEM (complemented with glutamine, 10% Hyclone FetalLOne III serum, GE Healthcare and 1% Penicillin/ Streptomycin) was added to the solution to inhibit trypsin action, and cells were dissociated with a Pasteur pipette. The cells were passed through Cell Strainer, and cells were counted in a Neubauer chamber, and each Flask T75 (Sarstedt) was plated with 1x106 cells. The medium was changed every three days and kept for 10-14 days. The culture was plated in a 6-well plate or 24-well plates for experiments. The culture used for the experiments was considered an astroglial-enriched culture based on previous data showing 91,2% astrocytes labeled with GFAP (Kinoshita, Yshii et al., 2017). To obtain astrocyte-pure cell cultures (>98%) for NF-kB experiments, flasks were incubated in an orbital shaker at 37°C , 180 r/min, for 15 h as previously described (Mazucanti, Kawamoto et al., 2019).
Primary cortical neuronal culture was prepared from both male and female postnatal (P1-3) mice C57BL/6 (Mus musculus). The meninges were separated from the brain, and the cortex were cut into small pieces and incubated in trypsin solution (2 mg/mL) for 20 min in a 37 ̵ͦC, 5% CO2 incubator. After removal of trypsin solution, tissue was washed twicce with HBSS.Tissue was dissociated in HBSS containing 0.1 mg/mLDNAse by mechanical trituration with glass pipette. Cells were counted and plate (1x106 cells) in polyethylene (Sigma-Aldrich) pre-coated dishes. Neuons were maintained for wwo weks in Neurobasal medium (GIBCO) supplement with B27 (GIBCO), 2mM gluatamine, 100 U/mL penicillin,100 mg/mL streptomycin and 0.25mg/mL amphotericin B
Both cell culture preparation (glial cells and neurons) were conducted in accordance with The Ethical Principle in Animal Research adopted by the Brazilian College of Animal Experimentation (CONCEA) and were approved by the Ethical Committee for Animal Research (CEEA) of the Biomedical Sciences Institute of the Universidade de São Paulo, São Paulo, São Paulo State, Brazil. The protocol was registered under number 12/2016 CEEA of animals used for experimentation.