a-Syn and Innate Immunity Figure 6. Effect of a-Syn-stimulation on microglial phagocytosis. (A) After treatment of the primary microglial cell cultures with a-Syn (1 mg/ ml) for 20 hours, cells were incubated with fluorescent microspheres for 1 hour. After fixing the cells, phagocytosis was assessed by fluorescence microscopy analysis. The phagocytic index was calculated by dividing the fluorescence from the phagocytosed microspheres by the total number of cells in the images. Four images were analysed for each sample in each experiment, and the results shown are representative of three independent experiments. A.U.: arbitrary units. (Representative microscopy images used to determine the phagocityc activity of microglial cultures stimulated with Wt (top) an A30P (centre) a-Syn, or non stimulated microglial cells (bottom). From left to right, green fluorescent microspheres, Hoechst-stained cells, and cells as observed in the absence of fluorescence. Scale bar: 50 mM. doi:10.1371/journal.pone.0013481.g006 against water. Afterwards, the protein preparations were concen- trated by centrifugation using a 10 k Amicon Ultra-Centrifugal Filter Device (Millipore Iberica, Madrid, Spain), and passed under sterile conditions through a 0.22-mm filter, and stored at 280uC. PAGE electrophoresis analyses of the a-Syn preparations Ten mg of protein were loaded onto a 15% SDS-PAGE gel (the loading buffer containing 0.1 M DTT), or 25 mg onto a 4–12% gradient Tris-glycine native PAGE gel (Lonza Group, Basel, Switzerland), and subjected to electrophoresis at 200 V. Gels were stained with Blue Silver [82], and destained with water. For Blue- Native PAGE (BN-PAGE), samples were prepared according to the protocol described previously for purified samples [83] with some modifications. Briefly, 12 mg of protein for experimental samples and 20 mg for protein markers were loaded on a continuous 15% poly-acrylamide gel. Ovalbumin and bovine serum albumin (Sigma-Aldrich, St. Louis, USA) were used as protein markers for native PAGE and BN-PAGE assays. After PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13481