Mem amino acid solution

Cortices from 1-day-old pups were extracted and placed onto a 100-mm petri dish using aseptic techniques. Cortices were sliced with a commercial razor blade, further broken up with a rigorous up-and-down motion in 10 mL of medium, and filtered with a 70-μm filter. The cells were then plated onto a 100-mm petri dish and put in an incubator of 37 °C with 5 % CO₂. Cell culture medium DMEM/F12 (Wisent, St. Bruno, QC) was supplemented with 10 % FBS (Invitrogen, Burlington, ON), 1 % penicillin-streptomycin solution (Wisent, St-Bruno, QC), 1 % L-glutamine solution (Wisent, St. Bruno, QC), 0.9 % sodium pyruvate solution (Wisent, St. Bruno, QC), 0.9 % MEM amino acid solution (Wisent, St. Bruno, QC), and 0.9 % amphotericin B solution (Wisent, St. Bruno, QC). The medium of the mixed glial culture was changed every 2 to 3 days. After 3 weeks, primary microglia cells were separated from astrocytes using EasySep CD11b positive selection kit following the manufacturer’s instructions (Stem cell, Vancouver, BC).

Brains were extracted and meninges removed from 1-day-old pups. Tissue was chopped and passed through a 70-μm filter. The cells were cultured in DMEM/F12 medium with 10% FBS (Invitrogen, Burlington, Canada) supplemented by 1% penicillin-streptomycin solution, 1% L-glutamine solution, 0.9% sodium pyruvate solution, 0.9% MEM amino acid solution, and 0.9% amphotericin B solution (all from Wisent, St. Bruno, Canada). The medium of the mixed glial culture was changed every 2 to 3 days. Primary glial cells were ready for experiments after 3 weeks. Glial cells were treated with LPS (100 ng/mL) and/or IFNγ (10 ng/mL) for 24 hours, and the conditioned media were collected and stored at −80°C.