All studies with animals were approved by the UIC Institutional Animal Care and Use Committee. Primary mixed glial cells were prepared from the frontal cortices of grouped male and female post-natal day 2 Sprague Dawley rats from the same litter [37]. In brief, cerebral cortices were cleaned from all meninges, digested in trypsin, and dissociated into single cell suspension by trituration through syringes. The cells were plated onto poly-l-lysine-coated flasks and grown in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% antibiotics (P/S; penicillin/streptomycin, Gibco, ThermoFisher, Waltham, MA, USA). The next day, cells were washed with PBS to remove debris, and the media were changed twice per week. After 7–10 days, loosely attached microglia were removed from underlying astrocytes by shaking flasks at 220 RPM for 30 min at 37 °C. Cells were collected, replated into dishes in DMEM containing 10% FBS and 1% P/S, and allowed to adhere overnight. The next day, the media were changed to serum free DMEM with 1% N-2 supplement.
