4% paraformaldehyde for 10 min at RT, washed with 10x volumes cold wash buffer, then permeabilized with PBS containing 17855348 0.1% saponin and 1% BSA for 15 min at RT, with shaking. Cell suspensions were transferred to FACS tubes, incubated with FITC-conjugated anti-human IL-8 or control human IgG, vortexed, incubated for 30 min at RT in the dark, then washed and fixed in 1% paraformaldehyde in PBS, and analyzed by flow cytometry. In all experiments, 10,000 events were collected from a large gate to exclude debris, but to include all cells. 518303-20-3 web intracellular ROS Production and Ca2+ Measurement To assess whether SAA and/or S100A12 altered intracellular ROS production in PBMC, we measured its levels by incubating cells with CM-H2DCFDA -chloromethyl-29,79-dichlorodihydrofluorescein diacetate, acetyl ester; Molecular Probes, Life Technologies), based on the manufacturer’s protocol. CMH2DCFDA is hydrolyzed intracellularly by esterases to H2DCF, a non-fluorescent and membrane-impermeable product, and subsequently oxidized to fluorescent DCF in the presence of ROS. PBMC were incubated for 30 min with 5 mM CM-H2DCFDA in HBSS containing Ca2+/ Mg2+, supplemented with 0.1% BSA and 10 mM HEPES at 37uC in 5% CO2 in air, then washed twice and incubated with the appropriate stimulus for 30 min. DCF fluorescence was analyzed by flow cytometry on cells gated on forward/side scatter profiles for the monocyte population; 5000 events were collected from a large gate that excluded debris, but included all cells. A second approach to investigate whether SAA altered intracellular ROS levels in monocytes, was to use diphenyleneiodonium, an inhibitor of NADPH oxidase, to reduce ROS generation. THP-1 cells were treated with SAA 6 DPI, and IL-8 levels measured by 9504387 ELISA. Ca2+ flux was determined with the Ca2+-sensitive fluorochrome using a PerkinElmer LS 55 fluorescence spectrophotometer as described using THP-1 cells. To measure i, cells in 2 ml were placed in a quartz cuvette with a magnetic flea in a Perkin-Elmer LS 55 spectrofluorimeter and stimulated with various concentrations of SAA. Ionomycin was used as a positive control to achieve maximum Ca2+ flux; EGTA was used to inhibit cells to estimate an approximate minimal response. Fluorescence was measured 
at 506 nm and 526 nm every 100 msec. Results expressed as relative fluorescence units at 506 nm over time. Superoxide dismutase levels in S100A126 SAA-treated THP-1 cells were determined based on inhibition of pyrogallol, using a 96-well microassay as described in. SDS-PAGE and Western Blotting To determine whether SAA and S100A12 formed complexes that may affect SAA function, S100A12 was cross-linked with SAA using bis suberate in the presence or absence of 1 mM Ca2+. Proteins were suspended in PBS and cross-linked for 30 min at RT in the dark, according to manufacturer’s instructions. Complexes were resolved on 10% SDS-PAGE gels under non-reducing conditions, then silver stained as described. To assess whether NF-kB and MAPK, CaMKII or SHP-1 signaling pathways were involved in S100A12 suppression of SAA-mediated cytokine production, time dependent IkB degradation and phosphorylation state of ERK1/ 2, MEK1/2, p38, JNK, CaMKII and SHP-1 were detected by Western blotting. After stimulation for the appropriate time, cells were washed once with cold PBS, then lysed in lysis buffer containing 50 mM Tris; pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 10% glycerol, 1% NP-40, 0.1% SDS, 0.5