of TUNEL positive nuclei were condensed or fragmented

Main astrocytes were prepared from the cerebral cortices of just one 3 day old Sprague Dawley rats as described by McCarthy and deVellis with minor alterations. Briefly, cerebral cortices were dissected and meninges removed. The tissues were minced and suspended Dasatinib in 10 volumes 0. 05-23 tryp sinEDTA and incubated for 10 min at 37 C. The cell suspension was passed through a 14-gauge needle 5 times, and then filtered through 85 mm nylon mesh. The filtrate was sedimented by centrifugation at 200 g for 5 min and re-suspended in 10 % FBS in DMEM con taining 100 unitsml penicillin and 100 ugml strepto mycin. Finally, cells were used in 75 cm2 culture flasks and fresh medium was changed 24 hours later and then every 2 days afterwards. Flasks were shaken at 200 rpm on an orbital shaker for 4 h at room temperature Plastid to remove microglial cells, when cells turned con fluent, normally within 7 9 times. After moving, cells were rinsed three times with phosphate buffered saline, stopped in trypsin containing solution as above, and subcultured in 12 well plates for Griess reaction experiment and 6 well plates for Western blot analysis. These countries included over 95% astrocytes, as based on immu nostaining for glial fibrillary acidic protein. For immunohistochemistry trials, astrocytes were cul tured on Poly M Lysine Coated Glass Coverslips. Cells were starved for 4 h prior to testing in serum free DMEM medium and followed closely by treat ments with different conditions as described. For preparation of primary microglial cells, rat or mouse pups less than 4 days old were used. The project TCID was much like that used for preparation of primary astrocytes. Quickly, after eliminating the meninges, brain tissue was minced into small pieces and trypsinized by incubating tissue at 37 C for 20 min. Brain tissue was triturated with a pipet to further dissociate sections and filtered with a 70 um cell strainer. Cells were centrifuged at 1,200 rpm for 5 min at 4 C, and pellet was suspended in 30 ml of complete medium containing DMEM with high glucose, 10 % FBS, OPI, and GM CSF to enhance prolif eration of microglia. The cell suspension was added to 75 cm2 flasks. Cells were incubated in flasks until confluent for 7 10 days. Microglial cells were separated from oli godendrocytes and astrocytes by shaking the flasks in a circular platform in a 37 C incubator at 200 rpm overnight. The superna tant, which was enriched with microglial cells, was then eliminated and centrifuged at 1200 rpm for 45 min. The microglia populace was established by immunostaining with CD11b antibody. Love for these microglial cells was determined to be around 95%. The cells were plated for experiments using complete media without the GM CSF. In most experiments, cells were serum starved for 4 h before adding cytokines and LPS. Cell morphology was observed using a phase contrast Nikon DIAPHOT 300 microscope connected with a CCD cool camera linked to MagnaFire 2.