Mean values were calculated from three separate experiments

Primary astrocytes were prepared from the cerebral cortices of 1 3 day old Sprague Dawley rats as explained by deVellis and McCarthy with minor changes. Quickly, cerebral cortices were dissected and meninges removed. The tissues were minced and suspended in 10 volumes Dapagliflozin 0. 05-22 tryp sinEDTA and incubated for 10 min at 37 C. The cell suspension was passed through a 14-gauge needle 5 moments, and then filtered through 85 mm nylon mesh. The filtrate was sedimented by centrifugation at 200 g for 5 min and re-suspended in ten percent FBS in DMEM con taining 100 unitsml penicillin and 100 ugml strepto mycin. Eventually, cells were used in 75 cm2 culture flasks and new medium was changed every 2 days the next day and then afterwards. When cells became con proficient, typically within 7 9 times, flasks were shaken at 200 rpm on an orbital shaker for 4 h at room temperature to eliminate microglial cells. After moving, cells were suspended in trypsin containing solution as above, Meristem rinsed three times with phosphate buffered saline, and subcultured in 12 well plates for Griess effect test and 6 well plates for Western blot analysis. These countries included more than 956 astrocytes, as determined by immu nostaining for glial fibrillary acidic protein. For immunohistochemistry findings, astrocytes were cul tured on Poly M Lysine Coated Glass Coverslips. Cells were starved for 4 h before analysis in serum free DMEM medium and accompanied by treat ments with various conditions as described. For preparation of major microglial cells, rat or mouse dogs significantly less than 4 days old were used. The protocol was similar to that employed for preparation of primary astrocytes. Quickly, SMER3 after removing the meninges, brain tissue was minced in to small pieces and trypsinized by incubating tissue at 37 C for 20 min. Brain tissue was triturated with a pipet to further dissociate clumps 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 total medium containing DMEM with one hundred thousand FBS, high glucose, OPI, and GM CSF to enhance prolif eration of microglia. The cell suspension was included with 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 rotary system in a 37 C incubator at 200 rpm overnight. The superna tant, which was enriched with microglial cells, was then removed and centrifuged at 1200 rpm for 45 min. The microglia citizenry was recognized by immunostaining with CD11b antibody. Purity for these microglial cells was determined to be around 95%. The cells were plated for experiments using complete media with no GM CSF. In most experiments, cells were serum starved for 4 h before adding LPS and cytokines. Cell morphology was observed by using a phase contrast Nikon DIAPHOT 300 microscope connected with a CCD great camera related to MagnaFire 2.