After washing three times with ice cold lysis buffer

The cells were grown at 37 C in moist 5% CO2 atmosphere, and the channel was repeatedly replaced Gemcitabine Cancer every 2 d. The mediwere replaced with serum free medi12 h before drug treatment. The cells were then treated with Abetor Abetfor 24 h. Epo at various concentrations were added into the cultures 1 h before the 24 h Abetexposure. 20 uM LY294002 were added to the cultures 1 h prior to the Epo treatment. Evaluation of cell viability Cell viability was assessed by MTT assay. Shortly, PC12 cells were seeded in 96 well culture dishes at density of 1 104 cells per well. After the cure of Abeta, Abeta, Epo or LY294002, the cells were subjected to the analysis as previously noted. Hoechst 33258 staining For Hoechst 33258 staining, cells were fixed with four or five par aformaldehyde. Mobile nuclei were stained with fluorescent dye Hoechst 33258 at remaining con centration of 5 ugml in PBS, for 20 min at room temperture in dark chamber, and then seen in fluorescence microscope and photographed. Western blotting The Western blotting analysis method was performed as previously described. Following the therapy, cells were washed Skin infection twice with cold phosphate buffered saline and lysed on ice with cell lysis buffer, 60 ugmL aprotinin, 10 ugmL leupeptin, 1 ugmL pepstatin for 30 mininutes. The soluble fraction was obtained by centrifu gation at 14000 g for 20 min at 4 C. The attention of the protein was based on the BCassay. Similar levels of the pro tein were separated within an 8 10 percent SDS polyacrylmide solution, the fixed proteins were electrotransferred onto PVDF or nitro-cellulose membranes. The walls were subsequently blocked with five minutes nonfat milk in TBST for 1 h at room temperature and incubated with 1,1000 for Cleaved caspase 3, 1,5000 for betactin, appropriate levels of primary antibody and Z-VAD-FMK 187389-52-2 PARP at 4 C over-night. The walls were then washed 3 times with TBST and probed with the corresponding secondary anti bodies conjugated with HRP at room temperature for 1 h. After washing, the signals were created using the ECL Advanced level Wes tern Blotting Detection kit. Group intensi ties were quantified by densitometric analysis by using an AxioCam electronic camerand the KS400 photo analysis program. Statistics Datare expressed as mean standard deviation and were analyzed using SPSS 11. 0 mathematical software. Each procedure was per shaped in duplicate in 3 5 separate experiments. Statistical analyses were done using one way ANOVA, followed by both tailed Students t test. Multiple comparison tests were applied when appropri ate, and statistical significance was thought at P 0. 05. Results Aftereffects of Abeton cell viability and cell apoptosis determined by Hoechst and MTT 33258 staining respectively The MTT assay was used to ascertain the aftereffect of 20 uM Abeton the viability of the PC12 cell cul tures.