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.