The stimulation was performed under serum free conditions. The conditioned medium was then collected, and separated by centrifugation at 400 g for 5 minutes to remove cell debris. The wells of micro titer plates were coated with conditioned medium over night. www.selleckchem.com/products/PF-2341066.html Plates were washed with PBS plus 0. 1% Triton Inhibitors,Modulators,Libraries X 100 and blocked with PBS plus 5% BSA for 1 hour. Plates were emptied, and any remaining liquid was tapped out onto dry paper towels. Rabbit polyclonal anti mouse PAI 1 antibody was added and incubated for 5 hours. Plates were washed three times with PBS T to remove unbound antibody. Horseradish peroxidase labeled anti rabbit IgG was added and incubated for 1 hour. Plates were washed three times with PBS T and developed by the addition of 100 ul of tetramethylbenzidene peroxide based substrate solution.

Inhibitors,Modulators,Libraries The recombinant mouse PAI 1 protein was used as a standard. Nitrite quantification Cells were seeded at the density of 5104 cellswell in 96 well plates, and treated with various stimuli for 24 hours in serum free medium. Production of NO was estimated by measuring the amount of nitrite, a stable metabolite of NO, using Griess reagent, as previously described. Assessment of cell viability and proliferation Cells were seeded in 96 well plates and treated with various stimuli for the specific time periods in Inhibitors,Modulators,Libraries the serum free medium. After treatment, 3 2,5 diphenyltetrazolium bromide assay was per formed as previously described. Microglianeuron co culture For the co culture of microglia and neurons, primary microglia were seeded at a density of 4104 cellswell in 96 well plates at 37 C in 95% air5% CO2.

After 16 hours of incubation, the cells Inhibitors,Modulators,Libraries were treated with LPS and mouse PAI 1 protein for 12 hours. Culture medium was then removed, and cells were washed with PBS. CMFDA labeled mouse primary cortical neurons were added to microglia plated wells and incubated in neurobasal medium containing 10% FBS. After an additional 24 hours incubation period, the number of CMFDA labeled cells was counted at 100 magnification in four visual fields in each well using a fluorescence microscope. Images of five random fields per well were captured and analyzed by an imaging system. Cell migration assays in vitro Cell migration was determined by using an in vitro scratch wound healing assay or Boyden chamber assay. The scratch wound healing assay was performed as pre viously described.

In brief, BV 2 mouse micro glial cells and C6 rat glioma cells were seeded at a density Inhibitors,Modulators,Libraries of 8104 cellswell in 96 well plates, and incu bated at 37 C under in 95% air CO2 for 14 hours. A scratch wound was created with a 200 ul pipette tip on the confluent cell monolayer. Cells were treated with or without pharmacological inhibitors, PAI 1 protein, BSA, RAP protein, Navitoclax Bcl-xL astrocyte conditioned medium, anti PAI 1 antibody, or rabbit serum. Cells were allowed to recover for 24 hours in serum free medium. The wound closure was then viewed under a microscope.