After changing the culture medium, microglia were added to these neuronal cultures with or without FGF 2 for 24 h. Cells were subsequently fixed in 4% paraformaldehyde. Microglia were stained with Cy5 conjugated rat anti this research mouse CD11b monoclonal Inhibitors,Modulators,Libraries antibodies prior to fixation. Phagocytic uptake of neuronal debris by microglia was estimated based on the detection of DiI stained neuronal debris in CD11b positive microglia . the phagocytosis index was calculated as the per centage of red staining that overlapped with green staining among all of the microglia. Immunocytochemistry Cells were fixed with 4% paraformaldehyde, blocked, and permeabilized. Neurons were stained with mouse poly clonal anti MAP 2 antibody and secondary antibody conjugated to Alexa 488.

Astrocytes were stained with mouse monoclonal anti GFAP antibody and secondary antibody Inhibitors,Modulators,Libraries conjugated to Alexa 647. Microglia were stained with Cy5 conjugated rat Inhibitors,Modulators,Libraries anti mouse CD11b monoclonal antibody prior to fixation. Images were analyzed using a deconvolution fluorescence microscope system. The other primary anti bodies included FGFRs, which were purchased from R D systems and used according to the manufacturers instructions. Surviving neurons were identified based on their cyto skeletons as previously described. Viable neurons were strongly stained with anti MAP 2 antibodies, whereas damaged neurons showed weaker staining. MAP 2 positive neurons were counted in representative areas in each well. Using five independent trials, more than 200 neurons were evaluated in each well by a scorer who was blind to the experimental conditions.

The number of viable neu rons in untreated cultures was set as 100%. Measurement of CCL3, NO, and glutamate levels Supernatants from microglia were assessed using the chemokine ligand 3 ELISA kit and a Griess reaction for nitric oxide detection. To measure glutamate levels, a colorimetric Inhibitors,Modulators,Libraries assay kit was used, as pre viously described. MTS assay To evaluate the viability of the cells, we used the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit and followed the manufacturers instructions. Microglial migration assay Microglial migration was performed using Transwell plates with 3 um pore polyethylene terephthalate membrane filters. We placed 800 ul of neuronal conditioned medium or microglial culture medium treated with drugs into the lower chamber of the Transwell plate.

Membrane filters were then put in vacant wells, and 200 ul of microglia containing medium was carefully added on top of the fil ter membrane to avoid bubbles. These plates were incu bated for 24 h. Cells that migrated Inhibitors,Modulators,Libraries into the lower wells were counted by fluorescence activated cell sorting. Chemokine treated T cells were used as positive controls for this method, as previously described. RT PCR Total RNA was extracted http://www.selleckchem.com/products/U0126.html from astrocytes, microglia, and neurons using an RNeasy Mini Kit.