Tissues were digested in 0. 25% Trypsin containing 0. 1% EDTA at 37 C for 15 min, cells were dis persed by gentle trituration, and seeded in Dulbeccos modified Eagles medium with 10% fetal bovine serum and 1% antibiotic solution in 75 cm2 T flasks at a den sity of 1 cortex flask. Cells were grown in the 37 C incubator kinase inhibitor MG132 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 at 1 �� 106 cells well 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 Inhibitors,Modulators,Libraries treatments LPS was selected as a control in this study due to its well established features as a potent pro inflammatory agent. Twenty four hours after re plating, mouse pri mary astrocytes were treated with fresh growth media containing 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 Inhibitors,Modulators,Libraries 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 Inhibitors,Modulators,Libraries inhibitor cocktail for 10 min on ice followed by brief sonication. Both media and Inhibitors,Modulators,Libraries 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 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 Inhibitors,Modulators,Libraries and cell protein extraction follow ing the treatments were harvested as above. Ab42 preparation and treatment Human Ab42 oli gomers and fibrils were prepared as previously described with minor modifications.
Briefly, Ab42 was dissolved in hexafluoroisopropanol, lyo philized, dissolved in dimethylsulfoxide to a concentration of 5 mM, and then diluted to make 100 http://www.selleckchem.com/products/Oligomycin-A.html 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.