Neuronal survival was determined by cell counting in 5 randomly selected phase contrast microscopic fields per culture well. Values were normalized to counts in control wells from the same 24 well plate. Regorafenib mechanism Microglia morphology was assessed by phase contrast microscopy of unfixed cells. Microglia with two or more thin processes were consid ered as ramified, resting microglia, Inhibitors,Modulators,Libraries and microglia with less than two processes, or with amoeboid cell soma, were classified as activated. The numbers of resting and activated microglia were counted in 5 randomly selected fields per culture well. Immunostaining was performed with cultures fixed with 1,1 methanol,acetone at 4 C. Cultures were characterized with antibodies to GFAP and Iba 1 as previously described. Antibody binding was visualized with suitable Alexa Fluor conju gated anti IgG.
Negative controls were prepared by omitting the primary antibodies. For detection of poly, cultures were incubated with rabbit anti body to PAR. Microglial phagocytosis of Ab was imaged using three dimensional confocal imaging of cultures with microglia astrocyte co cultures exposed to 5 uM of FAM Ab. Microglial phagocytic activity in microglial Inhibitors,Modulators,Libraries monocultures was quantified as described with minor modifications by measuring FAM fluorescence remaining in the cells after two washes with MEM. Nonspecific Ab adherence to the culture plate surface was evaluated by measuring FAM fluorescence in cell free culture wells that had been incubated with FAM Ab for 24 hours. Nitric oxide, cytokine and trophic factor measurements Microglial Inhibitors,Modulators,Libraries cultures were placed in 250 ul of MEM and incubated with Ab or rAb for 24 hours.
Nitric oxide production was measured by using Griess reagent as previously described. Cytokines and tropic factors were analyzed in 50 ul aliquots of cell culture medium using a Milliplex mouse multiplex immunoassay bead system according to the manufacturers Inhibitors,Modulators,Libraries instructions. Each sample was assayed in duplicate, and the fluorescent signal corresponding to each cytokine was measured with a BioPlex 200 system in parallel with known standards. Nonspecific interactions between beads and test compounds were screened by running the immunoassay with test com pounds dissolved in medium without cell culture expo sure. The reverse sequence Ab42 1 was found to interfere with the assay in a non specific man ner, and thus rAb treated cultures could not be ana lyzed.
Values for cytokine and trophic factor assays were normalized to the protein content of each well as deter mined by the bicinchoninic assay. Microglial NF B activity Microglia were infected with lentivirus encoding destabi lized, enhanced green fluorescence protein driven by the NF B promoter at 8 9 days in vitro, while Inhibitors,Modulators,Libraries still in co culture with astrocytes. considering Infection was performed in culture medium with viral titer of 6. 4 �� 10 8 pg of p24 antigen ml.