A standard curve prepared from recombinant TNF a was used to calculate the TNF a production of the samples. Nitric oxide determination in culture medium NO was measured as released NO metabolites using an NO detection kit. This such method uses nitrate reductase to specifically reduce NO3 to NO2. and the content of NO2 is determined colorimetrically. Briefly, 100 ul of incubation medium and a standard were added to the wells. Then, 50 Inhibitors,Modulators,Libraries ul of nicotinamide adenine dinucleotide and nitrate reductase was added. After 30 min, 100 ul of Greiss reagents I and II was added and incubated for 10 min at room temperature. The optical density of each well was determined using a microplate reader set at 540 nm. Flow cytometry Expression of microglial marker CD11b was mea sured by fluorescence activated cell sorting ana lysis to assess activation state of microglial cells.

Inhibitors,Modulators,Libraries Briefly, after 20 min of EMF or sham exposure, Inhibitors,Modulators,Libraries microglial cells were washed three times with flow buffer containing 0. 1% sodium azide and 1% BSA and re suspended in 250 ul of ice cold flow buffer. Cells were pre incubated with goat serum, Beijing, CN for 20 min at 4 C to block non specific binding to Fc receptors. Cells were then spun Inhibitors,Modulators,Libraries down at 5,000 rpm, washed three times with flow buffer, and incubated with rat anti mouse monoclonal antibody CD11b or rat IgG2b isotype control for 1 h at 4 C. Centrifugation and washing steps were repeated, and cells were then incubated with goat anti rat IgG DyLight549 for 1 h at 4 C in the dark. Quantitative analysis was performed using a FACSCalibur system.

Confocal microscopy with double label immunofluorescence As previously described, cultured cells were fixed and permeabilized. Cells were then pre incubated with goat serum for 20 min at room temperature and then washed 3 times with flow buffer. For immuno?uor Inhibitors,Modulators,Libraries escence labeling, cell cultures were incubated with one of the following antibodies for 1 h at 37 C rat anti mouse monoclonal antibody CD11b and rabbit anti mouse monoclonal pTyr705 STAT3 antibodies. For confocal microscopy of the double labeled samples, cell cultures were incubated simulta neously with goat anti rat IgG DyLight549 and sheep anti rabbit IgG FITC for 1 h at 37 C in the dark. Cell cultures were then washed and mounted with aqu eous based anti fade mounting medium. Images of stained cells were captured using a Leica TCS SP5 confocal laser scanning microscope.

Image analysis was performed with a semi quantitative method. Fluorescence intensity was measured using software Image J 1. 42. Western blotting Cells were washed with ice cold PBS and scraped in RIPA lysis buffer contain ing protease inhibitors. Whole cell extracts were separated by 8% SDS PAGE under reducing condi tions and Tipifarnib leukemia then transferred onto nitrocellulose mem branes. The membranes were blocked with a special Odyssey blocking buffer for 3 h at room temperature.