ml) was then added to the cells, and the plates were stored at 4uC for a minimum of 24 hours before being analyzed. Determination of the phagocytic index For each cell culture condition, the phagocytic capacity of microglial cells was determined by analysing fluorescent images of phagocytosed FITC-labelled microspheres and by staining cell nuclei with Hoechst 33342. An Olympus IX71 fluorescence microscope equipped with the digital image processing softwares DPController and DPManager (Olympus Europa, Hamburg, Germany) was used. For each random field, a mean phagocytic index was calculated by determining the intensity of specific green and blue fluores- cence emissions with digital imaging analysis software MetaMorph (MDS Analytical Technologies, Toronto, Canada). Specific green fluorescence emitted by FITC-microspheres was determined by subtracting the mean background fluorescence calculated from three different areas of the image where no cells were present, from the overall mean green fluorescence of the entire image. Specific blue emitted nuclear fluorescence was then calculated in a similar manner. The phagocytic index corresponds to the specific green/blue ratio. The mean phagocytic index was calculated from 4 random fields of cells (.100 cells) and was considered as a representative value of the phagocytic capacity of the microglial cells, as previously determined for mouse peritoneal macrophages within normal media for 24 hours with 1 ml/ml of lipopolysachar- ide (LPS). qRT-PCR Expression of GFAP and CD11b was determined by using a two-step quantitative real-time PCR. Total RNA from two microglial culture wells, one without treatment and one treated with LPS for 16 hours, was extracted using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufactur- er’s protocol. One mg of RNA was reverse-transcribed using the Quantitect Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocol. qPCR was performed with SYBRH Premix EX TaqTM (Perfect Real Time) (Takara Bio Inc., Otsu, Shiga, Japan) on an ABI Prism 7500 Real Time PCR System. Primers used were: HPRT_For: 59-GTAAT- GATCAGTCAACGGGGGAC-39, HPRT_Rev: 59-CCAGCAA- GCTTGCAACCTTAACCA-39; GFAP_For: 59-ATCGAGATC- GCCACCTACAG-39, GFAP_Rev: 59-CTCACATCACCACG- TCCTTG-39; CD11b_For: 59-CAGATCAACAATGTGACCG- TATGG-39, CD11b_Rev: 59-CATCATGTCCTTGTACTGC- CGC-39). Multiple transcripts were analyzed simultaneously for 40 cycles using an optimized qRT-PCR thermal profile. Changes in gene expression were determined using the 22DDCt method with normalization to endogenous hypoxanthinephophoribosyltransfer- ase (HPRT) control. Cytotoxicity assays The cytotoxic effect of the a-Syn variants under study was evaluated from the extent of LDH release with by using the LDH Cytoxicity Detection Kit (Roche, Basel, Switzerland) in MGC, following stimulation with the maximum concentration of wild- type a-Syn, its mutants or LPS (Sigma-Aldrich, St. Louis, USA), used in the present experiments (5 mg/ml for a-synuclein samples and 1 mg/ml for LPS). The limiting value in each case was References a-Syn and Innate Immunity determined using 6 mM MG132 (Sigma-Aldrich, St. Louis, USA), which is known to be lethal for cells at the concentration used, again using the manufacturer’s protocol. Cytokine release measurements Glial mixed cultures and isolated microglial cultures were stimulated with Wt a-Syn and its mutational variants at different concentrations (5 mg/ml and 1 mg/ml for MGC and 1 mg/ml and 0.2 mg/ml for MiG) for 20 hours. LPS at a concentration of 1 mg/ ml, and culture medium alone were used as positive and negative controls, respectively. Culture supernatants were harvested and centrifuged at 700 g for 5 min. and cell-cleared supernatants were recovered and stored at 280uC before cytokine measurement. IL- 6, IL-1b, TNF-a, IFN-c and IL-10 levels were assayed using Mouse IL-6/IL-1b/TNF-a/IFN-c/IL-10 BD OptEIA ELISA set (BD Biosciences, Madrid, Spain) according to the manufacturer’s protocol. Chemokine levels in the culture supernatants were determined by a specific sandwich ELISA by using capture/ biotinylated detection antibodies obtained from Peprotech (Lon- don, UK) according to the manufacturer’s recommendations. Cytokine profiles shown are representative of three independent experiments. Ethics statement All animals were handled in strict accordance with good animal practice as defined by the relevant national/EU guidelines and the CEA-CABIMER Experimental Animal Committee, and all animal work was approved by the appropriate committee (file CEA-2010-14). Data analysis All values are expressed as mean 6 S.E.M. Statistical significance (Student’s test, two-tailed) was evaluated using SPSS Statistics 17.0 (IBM Company, Chicago, USA). Supporting Information Figure S1. Time-course quantitation of total a-Syn in cell culture supernatants. Total a-Syn content measured by direct ELISA in culture supernatants recovered after addition of wild- type a-Syn to mixed glial cultures and incubation for 0, 1, 6 and 20 hours at 37uC. Found at: doi:10.1371/journal.pone.0013481.s001 (0.06 MB TIF) Figure S2. TNF-a and IFN-c release profile of a-Syn- stimulated primary microglial cultures. TNF-a (A) and IFN-c (B) levels were measured by ELISA in culture supernantants of microglia after a 20-hour treatment with exogeneously added a- Syn variants, or lipopolysaccharide (LPS). Values are mean 6 S.E.M. (n = 3). The results shown are representative of two independent experiments. Found at: doi:10.1371/journal.pone.0013481.s002 (0.13 MB TIF) Author Contributions Conceived and designed the experiments: CR CMD DP. Performed the experiments: CR ALG EGR RFM MC CCL CAW MD DP. Analyzed the data: CR ALG EGR RFM MC MD CMD DP. Contributed reagents/ materials/analysis tools: CAW. Wrote the paper: CR DP. 1. Spillantini MG, Crowther RA, Jakes R, Hasegawa M, Goedert M (1998) alpha- Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc Natl Acad Sci USA 95: 6469–6473. 2. Croisier E, Moran LB, Dexter DT, Pearce RK, Graeber MB (2005) Microglial inflammation in the parkinsonian substantia nigra: relationship to alpha- synuclein deposition. J Neuroinflammation 2: 14. PLoS ONE | www.plosone.org 11 October 2010 | Volume 5 | Issue 10 | e13481