02% deoxyribonuclease I was added. The culture medium consisted of DMEM F 12 nutrient mixture supplemented with 10% FCS, 0. 1% penicillin Pancreatic cancer streptomycin, and 0. 5 ug mL amphotericin B. Cells were pelleted, re suspended in culture medium, and brought to a single cell suspension by repeated pipetting followed by passing through a 105 um pore mesh. Cells were seeded at a density of 3. 5 �� 105 cells ml and cultured at 37 C in a 5% CO2 humidified atmosphere. Medium was replaced every 5 to 7 days. Microglial cul tures were prepared by the mild trypsinization Inhibitors,Modulators,Libraries method previously described by Saura et al. Briefly, after 19 to 21 days in vitro, mixed glial cultures were treated for 30 minutes with 0. 06% trypsin in the presence of 0. 25 mM EDTA and 0. 5 mM Ca2.

This resulted in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving a population of firmly attached cells identified as Inhibitors,Modulators,Libraries 98% microglia. The microglial cul tures were treated 24 h after isolation by this procedure. Experiments were carried out in accordance with the Guidelines of the European Union Council, Inhibitors,Modulators,Libraries following the Spanish regulations for the use of laboratory animals, and approved by the Animal Ethics Committee of the Universidad de Valladolid. Cultures were found to be 99% microglia by staining with FITC conjugated Griffonia simplicifolia lectin I B4 isolectin, a lectin that recognizes microglia, and an antibody against glial fi brillary acidic protein, to identify astrocytes. Primary and immortalized microglial cells were serum starved 24 h before the experiments, and then were stimulated for different times, as indicated, in the presence or absence of inhibitors.

Proliferation assay Cell proliferation was quantified Inhibitors,Modulators,Libraries using the Promega kit, Cell Titer 96RAqueous One Solution Cell Proliferation Assay values, as an assessment of the number of metabolically active cells. Microglia cell viability was also assessed by trypan blue exclusion. Western blot analysis After treatment, Inhibitors,Modulators,Libraries cells were washed twice with PBS and har vested in Laemmli SDS sample buffer. Protein extracts were separated by SDS PAGE and transferred to polyvinylidene difluoride membranes, which were incubated for 18 h at 4 C with the indicated antibodies, including ERK 1 2, p ERK1 2, p P70S6K, p rS6, COX 2 and actin. After washing with Tris Tween buffered saline, a 1,2. 000 di lution of horseradish peroxidase labeled immunoglobulin was added at room temperature for 30 h. The blots were developed using free overnight delivery enhanced chemiluminescence. Flow cytometric analysis BV 2 cells, 5 �� 106 flask, were treated with 1 ug ml of sPLA2 IIA for different periods of time at 37 C. Cells to be analyzed for expression of epidermal growth factor receptor were fixed in a mixture of 4% parafor maldehyde and 0.