For 10 min. The absorbance at 570 nm parthenolide (-)-Parthenolide was determined using a microplate Leseger t. The intracellular Re ROS was determined essentially as described previously. Briefly, PC12 cells seeded at a density of 1.5 9105 per well in 24 well plates t and at 37 ° C for 24 h after treatment with drugs, the cells were incubated with dichlorofluorescin diacetate 20.70 20 lm in serum-free MEM medium at 37 ° C loaded for 30 min. The above the Strength DCFH DA by three washes with Hanks Balanced Salt-L Solution removed. The cells were lysed with 1% Tween 20 at 1 M Tris buffer. The cell lysates were transferred into a 96-well fluorometric. The fluorescence was measured on a Packard fusion protein alpha fluorescence microplate Leseger t. ROS levels were expressed as a percentage of a contr On. The determination of intracellular Ren intracellular GSH content Rer GSH was determined by a modified HPLC as described above. At the end of the drug Sen treatment, cells were by trypsin / EDTA detached St and 1 ml ice-cold trichloroacetic in Acid. The mixtures were incubated on ice using a digital Ultraschallger t with 40% power for 60 s treated with ultrasound. The lysed cells were centrifuged at 6.8009 g for 1 minute. The whichever type were Walls min mixed with DTNB in 0.15 M phosphate buffer at room temperature for 10 min. After addition of 50 ll of 36.5% HCl, the samples were centrifuged at 6.8009 g for 1 minute. The whichever type Walls were applied to the HPLC system consisting of two Waters 626 LC pump, a Waters 717 plus sample injector, a Waters 996 photodiode array detector model, a contr The water content and a generator system Bergenin model 600S gradient analyzes. A column HP AlltimaTM C18 column coupled to a guard of all GuardTM is generated by a gradient elution of a mobile phase of the w Ssrigen L Solution containing 0.9% formic Acid and acetonitrile. L Solvent B was initially Highest 0-30% in 9 min increased Ht min, then 80% in 7, and min at 80% for a further fifth The flow rate was set at 0.8 ml / min. UV absorption at 326 nm was measured to detect GSH-conjugate. Detection of DNA fragments produced DNA fragmentation in apoptotic cells were characterized by agarose gel electrophoresis, as described above.
After treatment with drugs, PC12 cells were in 500 ll lysis buffer containing 0.2% Triton X-100, 10 mM Tris-HCl, pH 7.4, ethylenediaminetetraacetic-Acetic acid Acid 10 mM lysed on ice for 30 min. The whichever type Walls were coated with 100 lg / ml RNase A at 37 ° C for 1 h. The cellular Ren DNAs were using a standard procedure with extraction with phenol / chloroform / isoamyl alcohol and then End F Precipitation with 5 M sodium chloride and 100% ethanol isolated at 20 ° C overnight. DNA fragments were in 1.8% agarose gel containing 0.5 lg / ml ethidium bromide gel St. The gels were examined and photographed under UV light. Flow cytometric analysis of cell apoptosis as markers of apoptosis, the cell surface Surface phosphatidylserine by annexin V-FITC detection kit identified for apoptosis. Briefly, the cells were washed with PBS 19 and 19 re-suspended in binding buffer with a density from 1 to 9106 cells per ml. Cell suspensions were ll with 5 of annexin V-FITC and 10 ll of propidium iodide, resuspended and incubated at room temperature for 10 min. After removal of excess fluorescent agents, the task.