Two hundred microliters of each Streptozotocin 18883-66-4 cell lysate supernatant was mixed with 400 l Thiobarbiturs Acid reagent added. The reaction mixture was incubated at 37 in a shaking water bath for 1 h and then boiled for 30 min at 95. Then, the R Hrchen in a bath of zersto Transferred enem ice and centrifuged at 2500 g for 10 min. The amount of malonaldehyde was formed in each sample, with a 1,1,3,3 tetraethoxypropane standard curve dilution series by measuring the absorbance of the supernatant at 532 nm using a spectrophotometer against a blank. TBARS values are expressed as nmol MDA / h / mg protein. Oxidative modification of proteins was mixed with 2, 4 dinitrophenylhydrazine derivatization of oxidized proteins by the method of Reznick and Packer and Levine et al .. In general, cell protein in phosphate buffer 50 mM potassium phosphate lysates, pH 7.4, derivatized with DNPH. The samples contr The incubated with 2 N HCl at 1 h in the dark were mixed, the protein with 20% trichloroacetic Acid executed Was filled. Ethyl acetate: Derivatized proteins were washed with a mixture of ethanol. Final protein pellets were dissolved in 6N guanidine hydrochloride St and the absorbance at 370 nm. Protein carbonyl content is expressed in terms of moles / mg protein. The reverse transcriptase polymerase in each No RT-PCR was used to drive expression of iNOS mRNA, antioxidant enzymes and analyze NF . Macrophages Imatinib CGP-57148B were seeded in 24-well flat bottom plates T, and then preincubated with BP5 for 2 h, followed by 18 h with or without LPS.
RNA isolation was performed with an RNA extraction kit RNeasy based on the procedure performed by the manufacturer. The RNA was reverse transcribed into cDNA of 2 g of total RNA using a reverse transcription system from Promega. Separate, but simultaneous PCR amplifications were mixed with aliquots of cDNA to a final concentration of 1 PCR buffer, using 4 mM MgCl 2, 400 M dNTP and 2 U Taq DNA polymerase in a total volume of 20 l each of an L fwd rts and Rev rts primers. The primer sequences were hlt for iNOS according to claim Park et al selected .. The sequences of GPx, GR, Cu / Zn-SOD, Mn-SOD and CAT were hlt for Ferret et al selected .. The sequences of the p65 and NF Actin were hlt by Cohen et al selected .. Lahav The number of cycles was optimized uct and to product accumulation in the exponential area to weight. The PCR products of the respective cDNA were analyzed by electrophoresis on a 2% agarose gel and documents the Gel Doc EQ system. All signals were normalized to mRNA levels 饪 household Actin, and as a Caspase Pathway ratio Expressed ratio. iNOS activity t iNOS activity t was using a commercial assay kit. INOS activity Ts-assay is based on the F Ability of the synthase to catalyze arginine to NO, which further react with nucleophiles substances can k To connect chromophore which form a maximum absorption at 530 nm to produce. iNOS activity can t be determined on the basis that eNOS and nNOS are Ca 2, w while iNOS is independent ngig second Ca INOS activity t unit here as the amount is capable of 1 nmol NO in 1 min per milliliter of the medium defined. Total glutathione, GSH and GSSG were measured spectrophotometrically by the method of commercial test kit. Briefly, T GSH measured using the 5, 5 to dithio.