5 M, 2 10?2 M and 10?2 M, respectively, and were diluted selleck chemicals llc appropriately with cell culture medium. For in vivo studies, DON and SCM 198 were dissolved in 0. 9% sodium chloride so lution containing 1% sodium carbo ymethylcellulose. Lyophilized AB1 40 was first dissolved in sterilized distilled water followed by dilution with calcium free PBS to a final concentration of 1 mg ml. This solution was aggre gated at 37 C for 7 days before its application in in vitro e periment or in the surgery. Cell culture Cerebral corte of newborn SD rats was separated and cut into small pieces after removing meninges and blood vessels to prepare mi ed glial cells. Trypsinization with 0. 125% trypsin was stopped with DMEM F12 medium containing 10% FBS, 100 units ml penicillin, 100 ug ml streptomycin and 5 ug ml plasmo cin.

The tissue was gently pipetted to obtain a single cell suspension, which was then transferred to a new centri fuge tube after standing at room temperature for one to two minutes. This procedure was repeated three or four times. Then cells were centrifuged at 200 g for 5 minutes, resuspended in fresh DMEM F12 medium and plated according to different protocols. Twenty one days later, microglial cells were purified by mild trypsiniza tion method. For primary astrocyte culture, cortical mi ed glial cells from SD rats were cultured for two weeks. When cells became confluent, astrocytes were purified by shaking at 350 rpm at 37 C for 12 hours. The purity of primary microglia and astrocytes were confirmed with a mouse monoclonal CD11b antibody and a mouse mono clonal glial fibrillary acidic protein antibody, respectively.

Cerebral corte from fetuses of 17 to 18 days of gesta tion was used to prepare neurons, as described previ ously with minor modifications. Preparation of single cell suspension of neurons was the same with that of mi ed glial cells. Cells were maintained in neurobasal medium supplemented with 2% B27, 0. 5 mM L glutam ine, 100 units ml penicillin and 100 Anacetrapib ug ml streptomycin. Medium was changed 24 hours after plating and every three days thereafter. Neurons cultured for 10 to 14 days were used in the e periments. The purity of neurons was confirmed using a rabbit polyclonal MAP2 antibody. Immortalized murine BV 2 microglial cell line was first generated by Blasiet al. and retains many morpho logical and functional properties of primary microglia.

Cells were maintained in DMEM supplemented with 10% FBS, 100 units ml selleck compound penicillin and 100 ug ml streptomycin, and were passed twice a week. Microglia neuron co culture Microglia neuron co culture assay was performed as ac cording to Yuekui Li et al. with minor modifica tions. Neurons and BV 2 cells were separately seeded into 24 well or 6 well format transwell plates. BV 2 cells were pretreated with or without 0. 1 to 10 uM SCM 198 or 100 uM IBU for 2 hours and were stimulated with 1 ug ml LPS for another 2 hours.