Monocyte derived selleck inhibitor macrophage cultures Monocytes were obtained from the Center for AIDS Research of the University of Pennsylvania School of Medicine. The Center has IRB approval for blood collec tion from healthy donors. Monocytes were isolated by elutriation, the purity of isolated monocytes is higher than 95%. Blood samples were screened for common blood born pathogens and certified to be pathogen free. Inhibitors,Modulators,Libraries Freshly isolated monocytes were resuspended in DMEM supplemented with 10% fetal bovine serum, peni cillin, streptomycin and 1% non essential amino acids. Cells were cultured in 48 well plates at 2. 5 �� 105 cells per well. The medium was half changed every two days. After culture for 7 days, monocytes differentiated into macrophages.

Macrophages were first incubated with or without BBI for 24 h and then further treated with LPS for additional Inhibitors,Modulators,Libraries 24 h. Supernatants collected from the cell cultures were used to treat rat cortical neurons. Macrophages were lysed in 0. 5 mL Tri reagent for total RNA extraction. Assessment of neurotoxicity Neurotoxicity was examined Inhibitors,Modulators,Libraries by a cell based ELISA method which has been successfully used for measuring macrophage mediated neurotoxicity. Briefly, rat cortical neurons cultured in 96 well plates were treated with supernatant from LPS and or BBI treated macrophage cultures. To block the neurotoxicity of N methyl D aspartic acid, neural cultures were pretreated with 5 methyl 10,11 dihydro 5H dibenzo cyclohepten 5,10 imine maleate for 1 h. After 24 h treatment, cells were washed with PBS containing Ca2 Mg2 and then fixed in 4% paraformaldehyde 4% sucrose for 1 h at room temperature, followed by 1 h blocking in Block A.

Cells were then Inhibitors,Modulators,Libraries incubated with mouse mono clonal anti MAP 2 antibody diluted in block A overnight at 4 C. After a wash with PBS, goat a mouse b lactamase TEM 1 conjugate was added into each well and incubated for 30 min and then with fluorocillin green substrate solution in PBS for 1 h. Fluor escence was read at 485 527 nm in a fluorescence microplate reader. The fluorescence of untreated neurons was defined as 100%. Immunofluorescence staining Rat cortical cells were seeded on poly L lysine coated cover slips in 24 well plates and cultured for two weeks before treatment with supernatant from LPS activated macro phage culture. After treatment, cells were washed with PBS three times and fixed in ice cold methanol for 5 min.

Non specific sites were blocked in Block A for 30 min. Cells were then incubated in mouse Inhibitors,Modulators,Libraries anti MAP 2 antibody for 1 h, followed by Alexa 488 conjugated anti mouse IgG for 30 min. After Hoechst staining, the coverslips were mounted on glass http://www.selleckchem.com/products/AZD2281(Olaparib).html slide and observed under a fluorescence microscope. Reactive oxygen species detection Macrophages were pretreated with or without BBI for 24 h and then incubated with LPS for an additional 24 h.