Béraud et al. α-Synuclein andTLRs imaging. Images were acquired in tapping mode, using silicon tap- ping mode probes and a Multimode SPM Nanoscope IIIa system (Veeco/Digital Instruments, Santa Barbara, CA, USA). Nominal spring constants of 60 N/m and a resonant frequency of 245 Hz were used. EclErtluc BV-2 microglia treatment BV-2 murine microglial cells (BV-2 cells) were plated at a density of 5 × 105 cells per well (6-well plates) in 2 ml of DMEM sup- plemented with 5% fetal bovine serum and allowed to adhere for 24 h (Blasi et al., 1990; Horvath et al., 2008; Henn et al., 2009). One hour prior to treatment, serum-containing media was replaced with serum-free DMEM. Cells were subsequently treated with 50 nM SYNTR or BufferTR in DMEM for various time points as indicated in the figure legends. Following treatment, media were collected, centrifuged at 1000 rpm for 2 min and stored at −20°C until assayed. All treatments were preformed in triplicate in three separate biological replicates. TNF- α secretion Tumor necrosis factor-α levels in the media of treated microglia were measured by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). All measurements were performed in triplicate in three separate biological replicates. Nitric oxide release Nitric oxide (NO) release into the media of treated microglia was determined by measuring the stable NO metabolite, nitrite, using a Greiss reagent assay kit according to the manufacturer’s instruc- tions (Invitrogen). All measurements were performed in triplicate in three separate biological replicates. Preparation and treatment of primary microglia Primary microglia cultures were prepared from P1 to P3 C57Bl/6 mouse cortices as previously described (Su et al., 2008) except that microglia were isolated from mixed glial cultures (∼DIV 14) by shaking at 125 rpm for 5 h at 37°C on a rotary shaker and collecting the microglia-enriched medium. Microglia were plated at a density of 5 × 105 cells per well (6-well plates) in 2 ml of MEM supplemented with 0.01% pyruvate, 0.6% glucose, and 5% fetal bovine serum (microglia growth media) and allowed to adhere for 24 h. Cells were subsequently treated with 50 nM SYNTR or BufferTR in microglia growth media for 24 h. All treat- ments were preformed in triplicate in two separate biological replicates. Animals were maintained and treated in accordance with the regulatory standards of the Animal Welfare Act and approved for use by the Georgetown University Animal Care and Use Committee. qRT-PCR RNA was reverse transcribed in a 20 lμ reaction using a High-Capacity cDNA Archive Kit (Applied Biosystems, Carlsbad, CA, USA). The quality of cDNA was verified following RT-PCR for β-actin expres- sion. cDNA samples (10 lμ) were then added to 90 lμ of TaqMan ® Universal PCR master mix and loaded onto TaqMan® low density arrays (TLDA) preloaded with probes and primers for various targets and one endogenous control (see figure legends and Tables 1 and 2). Additional gene targets were assayed in a 96-well plate format, where 2.5 lμ of cDNA from each sample was added to 17.5 lμ of master mix containing the appropriate primer/probe pairs and TaqMan® Universal PCR master mix. All real-time PCR were run using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The results were analyzed using the relative quantification C∆t method, normalizing samples to endogenous controls, either 18S rRNA (TLDA) or GAPDH (individual targets) followed by normalization to the appropriate BufferTR treated controls.All measurements were per- formed in triplicate in three separate biological replicates. (Primers/ Probes used: TNF-α Mm00443258_m1, IL-1β Mm0434228_m1, NfkB1 Mm0047361_m1,Aif1 Mm00479862_g1, TLR1 Mm0120884_ m1, TLR2 Mm00442346_m1, TLR3 Mm00446577_g1, TLR4 Mm00445273_m1,TLR6 Mm01208943_s1,TLR7 Mm00446590_m1, TLR9 Mm00446193, Myd88 Mm01351743, Scarb1 Mm00450234_ m1, Hmox1 Mm00516004_m1, Prdx1 Mm01621996_s1, 18S rRNA Hs99999901_s1, and GAPDH 4352339E.) Statistical analysis was per- formed by Student’s t-test on ∆ Ct values and the significance level was set at P < 0.05. Gene expression changes are graphically represented as fold change (2−C∆t ). Western blot analysis BV-2 cells were treated as described above. Following treatment, cells were washed with ice-cold PBS and lysed on ice in modified RIPA buffer (50 mM Tris–HCl pH 7.4, 1% (v/v) NP-40, 0.25% (w/v) sodium deoxycholate, 150 mM NaCl) supplemented with Protease Inhibitor Cocktail (for mammalian cells; Sigma-Aldrich). Cell lysates were subjected to gentle rotation for 20 min at 4°C then sonicated at 18 Hz (3 × 10 s bursts with 30 s rest between bursts). Lysates were cleared by centrifugation at 17,000 rpm for 10 min at 4°C. Cleared lysates (25 gμ) were treated as described in above and subjected to denaturing polyacrylamide gel electrophoresis (10%) followed by transfer to PVDF membranes (PerkinElmer). Membranes were blocked in TBST/NFDM [20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween, 5% (w/v) non-fat dry milk] followed by incuba- tion with mouse anti-heme oxygenase-1 primary antibody (HO-1; 1:1000; Abcam, Cambridge, MA, USA). Immune complexes were visualized on film following incubation with HRP-conjugated goat anti-mouse secondary antibody (1:2000; Chemicon) and Super Signal West Pico Chemiluminescent Substrate. Membranes were reprobed with α-tubulin (1:1000; Abcam), which served as the loading control. Densitometric analysis of digitized images was completed using the EC3 Imaging System (UVP, Upland, CA, USA). RNA extraction Following treatment, RNA was harvested from cultured cells using ItatsIlacIsylna s an RNeasy mini kit and on-column DNase I digestion according All statistical analyses were carried out using Graphpad Prism 5 to the manufacturer’s instructions (Qiagen, Valencia, CA, USA). (Graphpad Software Inc., La Jolla, CA, USA). An ANOVA was per- RNA purity was assessed using an Agilent 2100 bioanalyzer (Santa formed followed by Bonferroni’s post hoc test or Student’s t-test Clara, CA, USA) and concentration measured using a NanoDrop where appropriate. All data are reported as mean ± SD. P-values 1000 spectrophotometer (Thermo Scientific). ≤0.05 were considered significant. www.frontiersin.org June 2011 | Volume 5 | Article 80 | 3