Exosome free heat inactivated horse serum

Cortices were dissected from the brains of C57BL/6 neonatal mice at post-natal day 2 and the meninges were removed. The tissue was dissociated in astrocyte culture medium [10% exosome-free heat-inactivated horse serum (Cat#2605088; Life Technologies, Carlsbad, CA), 10% FBS (Cat# 10082-147; Thermo Fisher Scientific, Waltham, MA), 1 mM sodium pyruvate (Cat# 11360-070; Thermo Fisher Scientific, Waltham, MA), 2 mM L-glutamine (Cat# 25030-149; Thermo Fisher Scientific, Waltham, MA), 1x Pen/Strep (Cat# 15140-122; Thermo Fisher Scientific, Waltham, MA) in DMEM (Cat# 11960-040; Thermo Fisher Scientific, Waltham, MA)] by gentle pipetting before the cell suspension was transferred to a T75 culture flask with a total medium volume of 12 mL. The cells were cultured at 37°C, 5% CO₂ and medium was changed twice over the first week of culture and thereafter as needed based on medium coloration. Microglia were first observed in the culture medium after ~2 weeks. The medium was collected for isolation and analysis of microglia at day 22.

Brain slices were individually cultured in 1.2 mL brain slice culture medium at 35°C, 5% CO₂ in 6-well plates (Cat# 3516; Corning Life Sciences, Corning, NY), supported on 0.40 μm Millicell Cell Culture inserts (Cat# PICM03050; MilliporeSigma, Burlington, MA). Brain slice culture medium consists of 50% BME (Cat#21010046; Life Technologies, Carlsbad, CA), 15% exosome-free heat-inactivated horse serum (Cat#2605088; Life Technologies, Carlsbad, CA), 0.25x Hank's BSS (Cat#H4641; MilliporeSigma, Burlington, MA), 2 nM GlutaMAX (Cat#A1286001; Life Technologies, Carlsbad, CA), 0.5% glucose (Cat#G68769; MilliporeSigma, Burlington, MA), 1x Pen/Strep (Cat# 15140-122; Thermo Fisher Scientific, Waltham, MA), 1x N2 supplement (Cat#17502048; Life Technologies, Carlsbad, CA) made up with sterile distilled water. Exosomes were pelleted and removed from the horse serum by ultracentrifugation at 100,000 g for 18 h at 4°C. Brain slice culture medium was replaced thrice per week including one full medium exchange and two half medium exchanges (400 μL removed, replaced with 600 μL due to evaporation); hPDGF (Cat#100-13A; Peprotech, Rocky Hill, NJ) was added at 10 ng/mL final to the medium on each occasion. Timepoints indicated in weeks, with each week constituting 7 ± 1 days in culture with the exception of “1 day” which indicates 22–26 h in culture.