A 96 well plate coated with monoclonal antibodies against the oxidative Wortmannin 19545-26-7 phosphoryl ation complex I and IV were used according to manufacturers instructions. Complex I activity was measured by adding an assay solution and the oxidation of NADH was moni tored by measuring its decrease in absorbance at 450 nm in kinetic mode at 30 C for 2 h. Complex IV activity was measured by adding an assay solution and the oxidation of reduced cytochrome c was monitored by measuring its de crease in absorbance at 550 nm in kinetic mode at Inhibitors,Modulators,Libraries room temperature for 30 minutes. Assays for mitochondrial re spiratory enzyme activity were performed using a Multis kan Spectrum reader. At least duplicate determination was carried out on each tis sue sample.
Qualitative and quantitative analysis of DNA fragmentation Tissue samples from the hippocampal CA3 subfield were subject to qualitative and quantitative analysis of DNA fragmentation. After extraction of total DNA from hippocampal tissues, nucleosomal DNA ladders were amplified by a PCR kit for DNA ladder assay to enhance Inhibitors,Modulators,Libraries the detection sensitivity, and were separated by Inhibitors,Modulators,Libraries elec trophoresis on 1% agarose gel. To quantify apoptosis related DNA fragmentation, a cell death ELISA that detects apoptotic but not necrotic cell death was used to assay the level of histone associated DNA fragments in the cytoplasm. Pro teins from the cytosolic fraction of hippocampal sam ples were used as the antigen source, together with primary anti histone antibody and secondary anti DNA antibody coupled to peroxidase.
The amount of nucleosomes in the cytoplasm was quantitatively determined using 2,2 azino di sulfonate as the substrate. Absorbance was measured at 405 nm and referenced at 490 nm using a microti ter plate reader. Statistical Inhibitors,Modulators,Libraries analysis One way analysis of variance was used, as ap propriate, to assess group Inhibitors,Modulators,Libraries means, followed by the Scheff�� multiple range test for post hoc assessment of individual means. All values are expressed as mean standard error of the mean. A P value 0. 05 was taken to indi cate statistical significance. Results Strategies for biochemical analyses and pharmacological treatments As in our previous studies, we routinely carried out biochemical analysis separately on tissues collected from the ipsilateral and the contralateral hippocampal CA3 subfield. This allowed us to ascertain selleck screening library that results from those analyses were consequential directly to ex perimental temporal lobe status epilepticus and not in directly to KA excitotoxicity. Since seizure activity was activated bilaterally, test agents were also routinely microinjected into the bilateral hippocampal CA3 sub field to confirm that parallel results were obtained from CA3 areas on both sides.