Primary antibodies to detect phosphorylated and total MAPK (Cell Signaling, Danvers, MA, USA) were applied at the concentrations recommended by the manufacturers

Primary antibodies to detect phosphorylated and total MAPK (Cell Signaling, Danvers, MA, USA) were applied at the concentrations recommended by the manufacturers. factor is fibroblast growth factor-2 (FGF-2). Although FGF-2 does exist in the mammalian central nervous system (CNS), it is unclear whether FGF-2 influences microglial function. Methods The extent of FGF-2 release was determined by ELISA, and the expression of its receptors was examined by immunocytochemistry. The effect of several drug treatments on a neuron and microglia co-culture system was estimated by immunocytochemistry, and the neuronal survival rate was quantified. Microglial phagocytosis was evaluated by immunocytochemistry and quantification, and microglial migration was estimated by fluorescence-activated cell sorting (FACS). Molecular biological analyses, such as Western blotting and promoter assay, were performed to clarify the FGF-2 downstream signaling pathway in microglia. Results Fibroblast growth factor-2 is secreted by neurons when damaged by glutamate or oligomeric amyloid 1-42. FGF-2 CCR4 antagonist 2 enhances microglial migration and phagocytosis of neuronal debris, and is neuroprotective against glutamate toxicity through FGFR3-extracellular signal-regulated kinase (ERK) signaling pathway, which is directly controlled by Wnt signaling in microglia. Conclusions FGF-2 secreted from degenerating neurons may act as a help-me signal toward microglia by inducing migration and phagocytosis of unwanted debris. (DIV) 14 using the shaking off method, which has been described previously [29]. The purity of the cultures was 97 to 100% as determined by immunostaining for the Fc receptor. Cultures were maintained in DMEM supplemented with 10% fetal calf serum, 5?g/ml bovine insulin, and 0.2% glucose. Astrocytes were purified from primary mixed glial cultures by three or four repetitions of trypsinization and replating. The purity of astrocytes was greater than 95%, as determined by GFAP-specific immunostaining [30]. Measurement of FGF-2 levels Secreted FGF-2 from mouse primary astrocytes, cortical neurons, and microglia CCR4 antagonist 2 were measured using an ELISA kit (RayBiotech, Inc., Norcross, GA, USA). Neurons were treated with L-glutamate (20?M) or oA (5?M) for 6 to 24?h at 37C. Supernatants were then collected and assessed for FGF-2 levels. Western blotting Microglial cell lysates were boiled after the addition of sample buffer (1?M Tris-HCl, 20% sodium dodecyl sulfate (SDS), and 2.5% glycerol). Fifty micrograms of total protein were separated on a 5 to 20% Tris-glycine SDS-polyacrylamide gel and blotted onto Hybond-P polyvinylidene difluoride (PVDF) membranes (GE Healthcare UK, Buckinghamshire, UK). Membranes were blocked with 1% skim milk in Tris-buffered saline containing 0.05% Tween 20 for 1?h at room temperature. Primary antibodies to detect phosphorylated and total MAPK (Cell Signaling, Danvers, MA, USA) were applied at the concentrations recommended by the manufacturers. The secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare), which was used at a dilution of 1 1:1000. SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL, USA) was used according to the manufacturers instructions. The intensities of the bands were calculated using the CS Analyzer 1.0 (Atto Corporation, Tokyo, Japan). Wnt promoter assay HEK293T cells were seeded one day before transfection by FuGENE HD (Promega, Madison, WI, USA) with a luciferase reporter vector from the Cignal TCF/LEF Reporter (luc) Rabbit Polyclonal to PARP2 kit (Wnt promoter assay system), which was purchased from SABiosciences (Qiagen KK, Tokyo, Japan). After drug treatment, cells were lysed and luciferase reporter activity was measured using the Dual luciferase reporter assay kit (Promega) and a Wallac 1420 ARVOMX (PerkinElmer Japan, Yokohama, Japan). CCR4 antagonist 2 Evaluation of microglial phagocytosis A microglial phagocytosis assay was performed as previously described [25]. Briefly, primary mouse cortical neurons in 24-well plates were labeled on DIV 14 with 1?M CM-DiI (Molecular Probes), and treated with 20?M glutamate overnight at 37C. After changing the culture medium, microglia were added to these neuronal cultures (1:2 ratio for neurons to microglia) with or without FGF-2 for 24?h. Cells were subsequently fixed in 4% paraformaldehyde. Microglia were stained with Cy5-conjugated rat anti-mouse CD11b monoclonal antibodies prior to fixation. Phagocytic uptake of neuronal debris by microglia was estimated based on the detection of DiI-stained neuronal debris [31] in CD11b-positive microglia (green); the phagocytosis index was calculated as the percentage of.