Gent with 400 L Opti MEM was mixed dropwise incubated to the above DNA / Plus complex for 15 min. Lentiviruses 293T line producing cells was transfected overnight with the DNA / lipofectamine / Plus complex in Opti MEM with 5% CO 2 incubator at 37 C. At the n Next day the medium was replaced with fresh DMEM containing 2% Cuscutin Bergenin heat-activated serum of f fetal K calf serum and continued incubation at 37 replaced C. After 48 h after transfection, the whichever type Collected walls, clarified Rt and filtered through Millex HV 0th 45 m filter polyvinylidene difluoride. The whichever type Walls were to do by adding 10% PEG 8000, incubation at 4 C overnight with 12 h, and centrifuged at 1500 g for 10 min at 4 C concentrates for the transduction of lentiviral shRNA expression in target cells construct, the growth medium of target cells by Opti-MEM containing 8 g / ml polybrene replaced and appropriate amounts of lentiviruses.
The cells were incubated overnight at 37 C. bcr-abl The medium was replaced with normal growth medium of n Next day. The efficiency of the hammer was tested 72 hours after the transduction. Alternatively was 2-4 g / ml added to 48 h puromycin after transduction and puromycin-resistant pool of cells was used for further experiments. Index Calculation The combined effect of the combination of ABT 737 and CPT 11 at a fixed rate of interest has been using software as reported previously CalcuSyn. Statistical analysis The values shown represent the mean _ SD for triplicate experiments. The statistical significance of differences between experimental variables was determined using the Student t-test.
P 0 05 was considered statistically significant. Results 11 ABT 737 and CPT in collaboration to improve the treatment-induced cytotoxicity t and apoptosis HCT116 cell line HT 29 with ABT 737 alone produced a modest reduction in the ability Lebensf Of the cells. In addition, coadministration of ABT 737 and CPT was shown 11 to the ability Lebensf Of the cells in a green Eren Ausma reduction than either drug alone. To determine whether the cytotoxic effect of the active ingredient combination is synergistic or additive, we performed an analysis using the average effective method. Lines HCT 116 and HT 29 cells were calculated with ABT 737 or CPT for 48 h and 11 of their IC50 values are treated.
The cell lines were treated with various concentrations of ABT 737 and CPT 11 in a fixed ratio Ratio and the Lebensf Ability of the cells was treated determined. The combination index was then calculated by the method of Chou and Talalay. As shown in an isobologram, were the CI values in HCT116 cells 1, which is a synergistic interaction. In HT 29 cells resulted in the calculation of the CI, that the combination of ABT 737 and CPT 11-additive. We analyzed and quantified the apoptotic effect of ABT 737 or CPT 11 alone and in combination with Annexin VF Staining. ABT 737, plus 11 CPT-induced apoptosis in a green Eren Ausma than either drug alone in both cell lines. ABT 737 induces apoptosis in a green Eren Ausma in HT 29 against HCT116 cells by the lower levels of endogenous Mcl 1 and h higher levels of Bcl-2 in HCT116 cells may be explained be rt. In addition, the drug combination produced a 1 7-fold in cell death compared to ABT 737 in HT 29 cells alone increased to 3 in comparison Ht. 5-fold increase in HCT116 cells. Improve ABT 737 and 11 CPT together apoptotic