Viable, apoptotic and necrotic cells were counted in 10 different fields under the 200�� vision in each well in three independent experiments by two persons and the average result was compared to the mock selleck chemical H89 control. Apoptotic cell numbers from different treatments were compared by being normalized to their viable cell numbers. Flow cytometry analysis of cell cycle HepB3 cells were selected to test the effect of miR 221 on cell cycle. Cells were plated into 6 well culture plates and treated with miR 221 inhibitor, mimic and their negative controls for 96h. Cells were collected with trypsin, then washed once with 4 C PBS, and fixed in cold 75% ethanol at 4 C. Cells were then washed once again with 4 C PBS and re suspended with PBS, then stained with 50 mg ml PI and 100 mg ml RNase A solution for 20 min at 37 C in dark.
Stained cells were subjected to analysis immediately by flow cytometry. The proportion of cells in each phase of the cell cycle was determined by a BDFACScan for Quantitative Cell Analysis. Caspase 3 7 activity detection Caspase 3 7 activity was measured using a synthetic rhodamine labeled caspase 3 7 substrate performed immediately after the detection of cell viability on the same wells, according to the instructions of the manufacturer. After incubation at room temperature for 60min, the fluor escence of each well was measured, using a FL600 fluorescence plate reader. Caspase 3 7 activity is expressed as fluorescence of treated sample mock control��100. Flow cytometry analysis of apoptosis HepB3 cells were also selected further to confirm the effect of miR 221 on apoptosis, using 7 Amino Actinomycin APC Annexin V with flow cytometry.
Cells were prepared as above and the procedure was according to the manufacturer. This assay allows to identify early apoptotic cells and late apoptosis or already dead. Statistical analysis SPSS19. 0 was used for statistical ana lysis. Results were representative of three independent experiments unless stated otherwise. Values were presented as the mean standard deviation. One way Analysis of Variance test and Students paired t test were used to analyze significance between groups. The Least Significant Difference method of multiple compa risons with parental and control group was applied when the probability for ANOVA was statistically significant. Statistical significance was determined at a P 0.
05 level. Background Up to 40% of patients with early stage breast cancer have disseminated tumors cells in the bone marrow. Fur thermore, bone is the most common site for breast cancer metastasis with 50% of metastatic breast cancer patients presenting with bone metastasis. Increased bone resorption is becoming increasingly recognized as a risk factor for development of metastatic tumor in the bone.