Cells were treated with or without pharmacological inhibitors, Oligomycin A clinical trial PAI 1 protein, BSA, RAP protein, 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. Relative cell migration distance was determined by measuring the wound width and subtracting this from the initial value, fold increase of migration distance. A total of three areas were selected and examined in each well. The fold increase of migration distance was based on the average wound width in three areas. The results were presented as the fold increase of the migration distance compared with control. A 48 well Boyden chamber was also used for the measurement of cell migration, in accordance with the manufacturers instructions.
Inhibitors,Modulators,Libraries The recombinant mouse PAI 1 protein in DMEM containing 10% FBS was placed into the lower wells, which were separated from the upper wells by polyvinylpyrrolidone free polycarbonate filters. Primary microglial cells were harvested by trypsinization, resuspended in serum free DMEM, and added to the upper chamber at a density of 5 �� 104 cells well. Cells were incubated at 37 C under in 95% air 5% CO2 for 24 hours. At the end of the incubation, any non migrating cells on the upper side of the membrane were removed with a cotton swab. Migrated cells on the lower part of Inhibitors,Modulators,Libraries the membrane were fixed in methanol for 10 minutes and stained with Mayers hematoxylin for 20 minutes. Photomicrographs of five ran domly chosen fields were taken, and cells were enumerated to cal culate the average number of cells that had migrated.
All migrated cells were counted. Results are presented as the mean SD of triplicates. Small interfering RNA transfection Control siRNA Inhibitors,Modulators,Libraries and mouse LRP1 siRNA pool were purchased from Santa Cruz Biotechnology. siRNA transfection of BV 2 micro glial cells was performed in accordance with the manufacturers instructions. The cells were harvested 48 hours after transfection, and used for the experiments. Dot blotting analysis Cells were treated with LPS, IFN, or mouse PAI 1 protein. Cells were then washed with PBS and lysed in triple detergent lysis buffer, 150 mmol l NaCl, 0. 02% sodium azide, 1% NP 40. Cell lysates were spot ted slowly onto nitrocellulose membranes.
The membranes were then blocked with 5% skim milk and sequentially incubated with anti LRP1 antibody and HRP conjugated anti rabbit IgG followed by ECL detection. Astrocyte conditioned medium To prepare ACM, primary astrocyte cultures were seeded at the density of 1. 5 �� 106 cells Inhibitors,Modulators,Libraries in 100 mm cul ture dishes. Primary astrocyte Inhibitors,Modulators,Libraries cultures were treated with a combination of LPS and IFN for 12 hours. Cells were then washed twice with PBS, and cultured Multiple myeloma in fresh DMEM for an additional 24 hours.