Immunocytochemistry Mice were transcardially perfused with ice cold PBS followed

The purity of primary microglial cells in the culture was assessed with staining of Iba 1 antibody ARQ 197 availability and Hoechst 33258. Cell viability Cell viability was determined by the tetrazolium salt 3 2,5 diphenyltetrazolium bromide assay. BV2 and primary microglial cells were initially seeded into 96 well plates at a density of 1 × 104 cells well and 5 × 104 cells well, respectively. Following treatment, MTT was added to each well and incubated at 37 C for four hours. The resulting formazan crystals were dissolved in dimethylsulfoxide. The optical density was measured at 570 nm, and results are expressed as a percentage of surviving cells compared with the control. Determination of cytokine production Medium TNF and IL 1B were measured using ELISA kits purchased from R D Systems following the manufacturers instruction.<br><br> Briefly, standards and samples were added to a 96 well ELISA plate precoated with biotinylated anti TNF or anti IL 1B antibody. AZD0530 ic50 After washing away unbound substances, an enzyme linked polyclonal antibody specific for TNF or IL 1B was added to the wells and incubated for two hours. The wells were then washed four times and filled with the substrate solution for an incubation of 30 minutes. The reaction was terminated by the stop solution. Absorbance was read at 450 nm in a microplate reader. The concentration of each sample was calculated from the standard curve prepared using the cytokine standards. NO release assay Medium nitrite was measured as an indicator of NO production. In brief, 50 ul of supernatant was mixed with an equal volume of Griess reagent I, followed by an addition of another 50 ul of Griess reagent II at room temperature.<br><br> Absorbance AMN-107 641571-10-0 was immediately measured at 540 nm. The samples were assayed in triplicate, and the concentration of each sample was calculated from a standard curve generated using sodium nitrite. PCR reaction was conducted as follows, an initial denaturation at 94 C for three minutes, 32 cycles of 94 C for 30 seconds, 48 C or 60 C for 45 seconds, 72 C for 30 seconds, then a final extension at 72 C for five was then blocked with 5% milk for one hour at room temperature. The membrane was incubated overnight at 4 C with primary antibody followed by a secondary horse radish peroxidase conjugated antibody for one hour at room temperature. Blots were developed using enhanced chemiluminescence according to the manufacturers protocol.<br><br> Primary antibodies against iNOS, p JNK1 2, p p38, p ERK1 2, p p65, JNK1 2, p38, ERK1 2, p65, and B actin, and secondary anti rabbit or anti mouse antibody were all purchased from Cell Signaling. PAR Microglia conditioned media Human SH SY5Y cells were plated in 96 well plates at a density of 1 × 104 cells per well and allowed to settle for 24 hours at 37 C before replacement with conditioned media. Culture media of BV2 cells with different treatments were collected as conditioned media and clarified by centrifugation at 20,000 × g for five minutes to remove cellular debris. The media were then transferred onto SH SY5Y cells. The viability of SH SY5Y cells was measured using the MTT assay as described above after 24 hours incubation. Statistical analysis Data were performed by a one way analysis of variance with Dunnetts test using the statistical package minutes.