Isolation and Conditioning of Neonatal Rat Astrocytes and Microglia

Primary neonatal rat astrocyte conditioned media was prepared as previously described [6 (link), 7 (link)]. For microglia conditioned media, primary neonatal rat glial cells were isolated and cultured as previously described [18 (link)]. Upon culture confluence, cells were dissociated using 0.25% Trypsin EDTA (Thermo Fisher Scientific) and microglia were isolated by fluorescence activated cell sorting (FACS) for CD11b/c positive cells. The sorted cells were cultured on poly-d-lysine coated plates (Sigma-Aldrich, 10 μg/mL) in DMEM with 10% FBS, and purity was confirmed by immunofluorescence and the absence of staining for astrocytes with a GFAP antibody. Conditioned media was collected after 48 h of incubation with microglia cultures of 70% or greater confluence. Mouse embryonic fibroblasts (MEFs) were purchased from ATCC and cultured in DMEM with 10% FBS. Conditioned media was collected after 48 h of incubation with the MEF cultures of 70% or greater confluence. All conditioned media were filtered through 0.4 μM filters prior to use to remove any dead cells or cellular debris.