Granulocyte monocyte colony stimulating factor

Primary murine microglia were isolated from postnatal day 0–2 pups according to previously published protocols [14 (link)] with a few modifications. Briefly, brains were isolated, meninges were removed, and were dissociated for 10 min at 37 °C with frequent agitation. Mixed glial populations were filtered through a 40-μm filter and plated in T75 flasks in DMEM/F12 supplemented with 20% heat inactivated fetal bovine serum (Sigma-Aldrich), 1% penicillin/streptomycin (Sigma-Aldrich), 1% l-glutamine (Sigma-Aldrich), and 10 ng/ml granulocyte monocyte colony stimulating factor (PeproTech) for 10–14 days. Microglia were isolated from the astrocyte bed by mechanical shaking at 195 rpm for 1 h at 37 °C and were counted and plated.

All animal protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Timed-pregnant C57BL/6J mice were obtained from Jackson Laboratories and P0-3 pups were isolated, meninges removed, and brain tissue dissociated and filtered (0.2 micron). Filtrate was plated into media containing 20% heat inactivated fetal bovine serum, penicillin/streptomycin, and L-Glutamine (Sigma), and 10ng mL^-1 granulocyte monocyte colony stimulating factor (PeproTech). Cells were maintained for two weeks and microglia isolated by mechanical shaking at 195 R.P.M. for 1 hour at 37C and plating to new wells.
For DQ Ovalbumin measurements, microglia were treated with fibrils or monomer for 30 minutes followed by the addition of DQ Green BSA (Molecular Probes) for one hour. Anti-iNOS (Abcam) and anti-MHCII (clone M5/114.15.2, eBiosciences) were used to stain microglia as previously described (Harms 2013). Images were captured with a Lecia TCS-SP5 confocal microscope and intensities were quantified using ImageJ software. Conditioned media was collected from each experiment and supernatants analyze with the Milliplex Mouse Cytokine/Chemokine Magnetic Bead 25 Plex Kit (MD Millipore)