Materials and Methods Sample preparation The proteins used in these experiments were recombinant S100A9 (MRP14) and Ab1-40 obtained from Alexo Tech AB(Umea, Sweden); Ab1-42 and Ab1-16 (Innovagen, Sweden). All experiments were performed with Ab peptide concentrations of 0.02 to 0.2 mM and S100A9 concentrations of 0.02 to 0.2 mM. S100A9 protein solutions were prepared in 10 mM Tris-HCl buffer at pH 7.4, and dialyzed to pH 3.0 in 20 mM Glycine buffer using Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, USA) at 40C. We dissolved Ab peptide at low temperatures (ice water) following a protocol released previously [24]. The chilled Ab peptide powder was initially dissolved in 10 mM NaOH at a concentration above 1 mg/ml and sonicated in an ice water bath for 1 min. Finally, a trace amount of 1 M NaH2PO4 buffer was added to adjust the sample to pH7.4 and 3.0. All other chemicals were purchased from Sigma unless mentioned differently. Amyloid kinetics Assay. A ThT stock solution was made by dissolving 2.5 mM Thioflavin-T (Merck-Schuchardt) in phosphate buffer (10 mM phosphate, 150 mM NaCl, pH 7.4) and filtered before use. This stock solution was diluted 50-fold in the phosphate buffer to generate the working solution. Protein samples of 10 mL of protein sample was taken added to 300 mL of the working solution, and was allowed to bind the ThT for 1 min. ThT Fluorescence was measured using a FluoroMax-2 spectrofluorometer (Jobin Yvon/Psex Instruments) with excitation and emission wavelengths of 440 and 48 5nm, respectively, and a slit width of 5 nm. The ThT fluorescence intensities were normalized to the fluorescence of the free dye in solution. CD spectroscopy. CD spectra were recorded with a JASCO J-720 spectropolarimeter equipped with a PTC-343 temperature controller. For each sample, 3 spectra were acquired and averaged in the range of 190 nm to 250 nm, using a 5 nm/min scan speed, and 1 nm resolution. The quartz cells had a 1 mm optical path. The contribution from the buffer was subtracted, and the results are presented in relative ellipticity. The difference ellipticity in Fig. 3 was obtained by subtracting the ellipticity of Ab1-40 peptide (0.002 to 0.1 mM) and S100A9 (0.2 mM) complex with the ones from 0.2 mM S100A9. Electrophoresis. SDS and native polyacrylamide gel electrophoresis (PAGE) was carried out on 15% Tris gels. 5 volume of protein sample was mixed with 1 volume of loading buffer (1.6 ml Tris-HCl (1 M), 4 ml SDS (10%), 2 ml glycerol (100%), 1 ml b-mercaptoethanol, 1.4 ml water and 4 mg Bromophenol blue; SDS and b-mercaptoethanol are excluded for native-PAGE). The electrophoretic separation was conducted in a Mini Protean II Electrophoresis system (Biorad, USA) with a running buffer (25 mM Tris and 192 mM glycine at pH 8.3; 0.3% SDS was added for SDS PAGE). The standard protein ladder was purchased from BioRad. Coomassie brilliant blue B7920 (Biorad, USA) (0.125 g Coomassie Brilliant Blue R-250 in 200 ml methanol, 35 ml acetic acid and 265 ml water, filtered before use) was used for staining, and the following destaining step was performed by using destaining agent (50% methanol, 10% acitic acid and 40% water) for around 2 hours with gentle agitation. Gas-phase electrophoretic mobility molecular analysis (GEMMA). The operation procedure of GEMMA instrument was as published previously [25]. S100A9 samples at pH 7.4 and 3.0 were exchanged against 20 mM ammonium acetate buffer (pH 7.4) by centrifugal ultrafiltration in Spin-X Centrifuge Tubes (Sigma-Aldrich, USA) before loading into the capillary tube. The operation current and voltage were approximately 300 nA and 2 kV respectively. Inflammation and Alzheimer’s Disease (AD) Atomic Force Microscopy (AFM). AFM measurements were obtained with a PicoPlus AFM (Agilent) in tapping mode using a 100 nm scanner in ambient conditions. Acoustically driven cantilevers with etched silicon probes of the TESP model (diameters of 10nm and less) (Digital Instruments) were used. We applied a resonance frequency in the 170 kHz to 190 kHz range, a scan rate of 1 Hz or less, and a resolution of 512 pixels6512 pixels. Height, amplitude and phase data were collected simultaneously in trace and retrace modes to avoid scan artifacts. Amyloid samples were deposited on the surface of freshly cleaved mica (GoodFellow) for 5 min, washed 3 times with 200 ml of DI water and dried in air at room temperature. Cell culture. SH-SY5Y (ATCC CRL-2266) were cultured in Dulbecco’s modified Eagle medium supplemented with 10%(v/v) FBS. Mixed cortical neuron cultures were prepared from embryonic day 16.5 rat embryos. Animals used in this study were maintained in strict accordance with the approval granted by Umea Ethical Committee for Animal Experimentation (Permit Number: A107–08). The cortical hemi-spheres were quickly dissected in ice-cold phosphate saline buffers (PBS, pH 7.2) with 10 mM D-glucose. Cortices from 6–8 embryos were pooled and neurons were isolated using the Papain Dissociation System (Worthington Biochemical Corp). Viable cells were counted following staining with 0.2% (w/v) trypan blue (Gibco/ Invitrogen) and plated at a density of 10,000 cells per well (100,000 cells/ml) in a 96-well black-walled imaging plate (BD Falcon). Neurons were cultured for 72 h in Neurobasal medium (Invitrogen) containing 2% (v/v) NS21 supplement (Chen et al. 2008), 0.5 mM L-glutamine, 100 units/ml penicillin and 100 mg/ ml streptomycin in a humidified incubator (370C/5% (v/v) CO2). WST1 cell viability assay. To evaluate cell viability, 10 ml of WST-1 reagent was added per 100 ml of cell culture and samples were incubated at 370C for 4 h. The absorbance was measured with an ELISA plate reader (Labsystem Multiscan RC) at 450 nm. Cell viability was expressed as a percentage of the absorbance in wells containing cells treated with amyloid compared to the control cells. Supporting Information Figure S1 Ab1–40 peptide (0.2 mM) incubated at pH 7.4 and 370C for (a) 1 d; (b) 3 d. The scale bars denote 600 nm and 2000 nm, respectively. The insertions show the cross-section profile of the fibrils corresponding to the marked positions. After 1 d incubation, proto-fibrils with a height of 0.5 nm were observed. After 3 d, the surface was dominated by fibrils with a height of 2 nm, which is typical for amyloid mature fibrils. As the fibrils observed upon the addition of S100A9 (Fig. 1) carried a height of 2 nm and the protofibrils are not necessarily related with amyloid formation, it is reasonable to conclude that the presence of S100A9 promotes the rate of formation and the quantity of Ab1–40 amyloid species produced. (TIF) Figure S2 CD septra (a) The transition in the secondary structures of (a) S100A9 (0.2 mM); (b) Ab1–40 peptide (0.2 mM). Ellipticity of S100A9 (0.2 mM) with the addition of various concentrations of Ab1–40 peptide (0.002 mM to 0.1 mM), incubated at pH 7.4 and 370C for (c) 3 d; (d) 7 d. The insertions denote the curves of 0.1 mM Ab1–40 in panel (c) and (d). (TIF) Figure S3 AFM height images of S100A9 (0.2 mM) (a) incubated at pH 3.0 and 570C with continuous shaking at 800 rpm for 3 d; (b) incubated at pH 3.0 and 370C for 3 d; (c) incubated at pH PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e32953