electrophoresis, gels were destained in a solution of 50% methanol and 10% acetic acid until the bands were clearly visible. Time-course determination of total a-Syn and oligomer content in the medium after incubation with cells For a quantitative assessment of the oligomeric content of culture supernatants, a specific sandwich ELISA assay [60] and a calibration curve with oligomers of an a-Syn variant carrying six point mutations (V63A, T64S, N65H, V66L, V71F, T72S), were used. Such oligomers were prepared as follows: a 700 mM solution in PBS of the mutant a-Syn was incubated overnight at RT, and subjected to centrifugation with a 100 kDa cut-off centrifugal filter (Millipore #UFC510024). The resulting retentate was rinsed with 150 ml of cold PBS and subjected to centrifugation as before. This procedure was repeated three more times, in order to eliminate the non-oligomerized protein. The oligomeric nature of the prepared a-Syn fraction was confirmed by native PAGE and Western blot (not shown), and the a-synuclein protomer concentration was determined with the BCA assay kit (Pierce, Rockford, USA.). Quantification of total a-Syn species in culture supernatants after 0, 1, 6 and 20 hours of incubation with cells at 37uC was performed by ELISA. Briefly: individual wells of 96-well ELISA plates were coated with 100 ml of cell-free culture supernatants. Plates were incubated at 37uC for 2 hours and washed with 0.5% Tween20/PBS, pH 7.2 (PBST). Plates were blocked with 200 ml of 1% BSA in PBS and incubation at 37uC for 1 hour. After washing with PBST, 100 ml/well of a 1 mg/ml biotinylated anti-a-Syn 211 mouse mAb [Santa Cruz Biotechnology, Santa Cruz, USA; biotinylated as previously described [60] diluted in 1% BSA in PBS were added and plates were incubated at 37uC for 1 hour. After washing with PBST, 100 ml/well of 1:5000 dilution of ExtrAvidin-alkaline phosphatase (Sigma-Aldrich, St. Louis, USA) in 1% BSA in PBS were added, and plates were incubated at 37uC for 30 min. After washing with PST, 100 ml/well of pNPP substrate (Sigma, St. Louis, USA) were added, and absorbance at 405 nm was measured within 30 minutes. Cell cultures Mixed glial cultures were prepared from the cerebral cortices of 1–3 days-old C57BL/6 mice (University of Seville Animal Core Facility, Seville, Spain) according to previously described methods [33] with some modifications. After mechanical, trypsin-mediated (BioWhittaker, Verviers, Belgium) dissociation, followed by filtra- tion in DMEM-F12 with 10% inactivated FBS (BioWhittaker, Verviers, Belgium), cells were cultured at 37uC onto 12-well plates treated with poly-D-lysine (Sigma-Aldrich, St. Louis, USA). After 2 days, half of the volume of culture medium was carefully changed, and completely changed after 4 days of culture. Cells were used for stimulation or microglial isolation at day 18–22 of culture. Microglial isolation was carried out according to previously described methods [84] with some modifications. The supernatant was removed and the wells were washed with DMEM-F12 without inactivated FBS; this conditioned medium was stored to be used later. Cells were incubated for 30–45 min with DMEM-F12/ trypsin 0.25% solution at 37uC and complete medium was added to inactivate trypsin and the supernatant containing microglial cells collected. Conditioned medium was added to attached microglia; the following day the medium was changed for normal medium, and microglial cultures were stimulated after 5 days post isolation. Immunofluorescence analysis Purified microglia were characterized by immunocytochemistry on the basis of their expression of the pan haematopoietic marker a-Syn and Innate Immunity CD45 and of the monocyte/macrophage markers CD11b, F4/80 and CD68. Additionally, the absence of Glial Fibrillary Acidic Protein (GFAP)-positive astrocytes in purified microglial cell cultures was also established. By contrast, trypsin-detached cells, largely astrocytes but containing some microglial cells, were plated back into 12-multiwell tissue culture plates and immunocyto- chemically processed as mentioned above. For detection of CD45, CD11b and CD68 cell-surface antigens, cells were fixed for 10 min at room temperature (RT) in PBS containing 4% paraformaldehyde, washed twice in PBS and finally blocked overnight at 4uC in PBS containing 3% bovine serum albumin (BSA). The cells were then incubated for 1 hour at 4uC with a fluorescein isothiocyanate (FITC)-conjugated rat anti- mouse CD45 monoclonal antibody (Leukocyte Common Antigen, Ly-5, clone 30-F11, BD Pharmingen, Franklin Lakes, USA) and a FITC-conjugated rat anti-mouse CD11b monoclonal antibody (clone M1/70, BD Pharmingen, Franklin Lakes, USA). Cells were exposed to primary rat anti-mouse F4/80 monoclonal antibodies (clone Cl:A3-1, Serotec, Oxford, UK) for 1 hour at RT at a final dilution of 1:100 in PBS. For detection of CD68 and GFAP intracellular antigens, 4% PFA-fixed cells were permeabilized and blocked overnight at 4uC in PBS containing 3% BSA and 0.5% of the permeabilizing detergent Triton-X-100. The following day, cells were incubated for 1 hour at 4uC in PBS containing a dilution of 1:100 rat anti-mouse monoclonal CD68 antibodies (clone FA-11, Serotec, Oxford, UK) and a 1:300 dilution of anti- GFAP mouse monoclonal antibodies (clone G-A-5, Sigma- Aldrich, Saint Louis, USA). Labelling with F4/80 and CD68 primary antibodies was detected from fluorescence measurements after incubating cells for 30 min at RT in PBS containing 1:100 diluted FITC-conjugated goat anti-rat IgG secondary antibodies (Jackson ImmunoResearch, West Grove, USA). For GFAP detection, an Alexa Fluor 488 nm goat anti-mouse IgG secondary antibody solution (Invitrogen, Paisley, UK) was added for 30 min at RT at a final dilution of 1:300 in PBS. Immunofluorescence images were captured with an inverted fluorescence microscope Olympus IX71 using the digital image processing softwares DP Controller and DP Manager (Olympus Europa, Hamburg, Germany). Phagocytosis assays FluoresbriteTM carboxylate 0.75 m microspheres (2.64% Solid- Latex; Polysciences Inc, Warrington, USA) were used as fluorescein-conjugated tracker microparticles for measuring the phagocytosis capacity of differentially activated microglial cells. One hour before starting the phagocytosis assay, fluorescent microspheres (1.0861011 particles/ml) were mixed at a ratio of 1 ml/20 ml inactivated FBS (BioWhittaker, Verviers, Belgium) and incubated for 1 hour at 37uC in order to opsonise fully the carboxylate groups. The mixture of microspheres and FBS was then resuspended in fresh DMEM-F12 medium (BioWhittaker, Verviers, Belgium), with L-glutamine and P/S antibiotics supplements to obtain normal 10% FBS-supplemented media containing 5.46108 microspheres/ml. After removal of 400 ml of supernantant from the a-Syn- stimulated microglial cell cultures, a volume of 150 ml of resuspended microspheres was added to each well to obtain a final concentration of 1.086108 particles/ml. The particles were then homogenously distributed throughout each well by gentle movements of the plate and incubated for 1 hour at 37uC. The medium containing non-phagocytosed microspheres was then removed and the cells were washed with PBS prior to their fixation with 4% paraformaldehyde in PBS for 30 min at 4uC. One ml of PBS containing the nuclear fluorescent dye Hoechst 33342 (1 mg/ PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13481