Cells were grown in the 37 C incubator with 5% CO2. After 12 days in vitro, the mixed glial cultures became a confluent monolayer, and cells were then detached by trypsinization and re plated Ganetespib cancer at 1 �� 106 cells well Inhibitors,Modulators,Libraries in 6 well plates for pro inflamma tory agent treatments. For Ab42 treatments, astrocytes were re plated at 5 �� 105 cells well in 12 well plates. The purity of astrocytes in the mixed glial cul tures with this method was verified using fluorescence immunocytochemistry by staining with anti glial fibril lary acidic protein and anti F4 80 antibodies. LPS and pro inflammatory cytokine treatments LPS was selected as a control in this study due to its well established features Inhibitors,Modulators,Libraries as a potent pro inflammatory agent.

Twenty four hours after re plating, mouse pri mary astrocytes were treated with fresh growth media containing Inhibitors,Modulators,Libraries pro inflammatory agents, either individually or in specific combinations at concentrations described previously. Single agent treatments were, LPS, TNF a, IL 1b, IFN g, combination treatments were, LPS IFN g, TNF a IFN g, TNF a IL 1b IFN g. After 24, 48, or 96 h of treatment, media were collected, cells were washed two times in ice cold D PBS, and then cells were lysed in buffer con taining 40 mM Tris HCl, pH 6. 8, 2% sodium dodecyl sulfate, 10% glycerol, 0. 02% sodium azide with freshly added protease inhibitor cocktail for 10 min on ice followed by brief sonication. Inhibitors,Modulators,Libraries Both media and cell lysate samples were stored at 80 C until analysis. Inhibitor treatments Inhibitors were prepared as concentrated stock solutions according to respective manufactures instructions.

The final concentrations Inhibitors,Modulators,Libraries of inhibitors in media applied to astrocytes were the following, 1400W, 1, 8, and 50 uM, JAK inhibitor, 1, 5, and 20 uM. Inhibi tors were added to culture medium 30 min prior to sti mulation of cells with TNF a IFN g for 96 h. Conditioned medium and cell protein extraction follow ing the treatments were harvested as above. Ab42 selleckbio preparation and treatment Human Ab42 oli gomers and fibrils were prepared as previously described with minor modifications. Briefly, Ab42 was dissolved in, lyo philized, dissolved in dimethylsulfoxide to a concentration of 5 mM, and then diluted to make 100 uM stocks either with ice cold phenol red free Hams F12 medium for making oligomers, or with 10 mM HCl at room temperature for making fibrils. Before treating cells, the 100 uM Ab42 stocks, and vehicle controls lacking Ab42, were incubated for 24 h on ice for oli gomers or at 37 C for fibrils. One day prior to Ab42 treatments, primary astrocytes were washed twice with D PBS, and changed into serum free media. Specifi cally, G 5 supplement was used at 1% to replace FBS in the growth media for primary astrocytes.